Compositions for reprogramming cells into dendritic cells or antigen presenting cells, methods and uses thereof

ABSTRACT

The present disclosure relates to compositions, nucleic acid constructs, methods and kits thereof for cell induction or reprogramming cells to the dendritic cell state or antigen presenting cell state, based, in part, on the surprisingly effect described herein of novel use and combinations of transcription factors that permit induction or reprogramming of differentiated or undifferentiated cells into dendritic cells or antigen presenting cells. Such compositions, nucleic acid constructs, methods and kits can be used for inducing dendritic cells in vitro, ex vivo, or in vivo, and these induced dendritic cells or antigen presenting cells can be used for immunotherapy applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 16/342,803 filed Apr. 17, 2019, which is a U.S. National Stage under 35 U.S.C. § 371 of PCT/IB2018/052378 filed Apr. 5, 2018, and which depends from and claims priority to Portugal Application No: 110012 filed Apr. 5, 2017, European Application No. 17171166.6 filed May 15, 2017, Portugal Application No 110263 filed Aug. 24, 2017, and Portugal Application No: 110267 filed Aug. 25, 2017, the entire contents of each of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 25, 2022, is named 2022-04-26_Sequence_listing_16 UCOI-H070303NA.txt and is 335,644 bytes in size.

TECHNICAL FIELD

The present disclosure relates to the development of methods for making dendritic cells or antigen presenting cells with antigen presenting capacity from differentiated, multipotent or pluripotent stem cells by introducing and expressing isolated transcription factors. More particularly, the disclosure provides methods for redirecting differentiated, multipotent or pluripotent stem cells to a dendritic cell or antigen presenting cell state by direct cellular reprogramming with a surprisingly use of combinations of transcription factors.

BACKGROUND

Cellular reprogramming relies on rewiring the epigenetic and transcriptional network of one cell state to that of a different cell type. Transcription factor (TF)-transduction experiments have highlighted the plasticity of adult somatic or differentiated cells, providing new technologies to generate any desired cell type. Through forced expression of TFs, it is possible to reprogram somatic or differentiated cells into induced pluripotent stem cells (iPSCs) that are remarkably similar to embryonic stem cells (1, 2). Alternatively, a somatic cell can also be converted into another specialized cell type (3). Direct lineage conversion has proven successful to reprogram mouse and human fibroblasts into several cell types, such as neurons, cardiomyocytes and hepatocytes, using TFs specifying the target-cell identity (4). Lineage conversions were also demonstrated in the hematopoietic system, where forced expression of TFs induced a macrophage fate in B cells and fibroblasts (5) and the direct reprogramming of mouse fibroblasts into clonogenic hematopoietic progenitors is achieved with Gata2, Gfi1b, cFos and Etv6 (6). These four TFs induce a dynamic, multi-stage hemogenic process that progresses through an endothelial-like intermediate, recapitulating developmental hematopoiesis in vitro (7).

Reprogrammed cells are very promising therapeutic tools for regenerative medicine, and cells obtained by differentiation of iPSCs are already being tested in clinical studies. For hematopoietic regeneration, however, approaches to generate mature blood cells from iPSCs are still lacking. Therefore, alternative strategies are needed to generate patient-specific definitive hematopoietic cells that can be used as blood products. Given the opportunity of direct cell reprogramming mediated by TFs, one can envision the generation of antigen-presenting cells (APCs) of the immune system such as Dendritic Cells (DCs).

DCs are professional APCs capable of activating T cell responses by displaying peptide antigens complexed with the major histocompatibility complex (MHC) on the surface, together with all of the necessary soluble and membrane associated co-stimulatory molecules. DCs induce primary immune responses, potentiate the effector functions of previously primed T-lymphocytes, and orchestrate communication between innate and adaptive immunity. DCs are found in most tissues, where they continuously sample the antigenic environment and use several types of receptors to monitor for invading pathogens. In steady state, and at an increased rate upon detection of pathogens, sentinel DC in non-lymphoid tissues migrate to the lymphoid organs where they present to T cells the antigens they have collected and processed. The phenotype acquired by the T cell depends on the context in which the DC presents its antigen. If the antigen is derived from a pathogen, or damaged self, DC receive danger signals, become activated and the T cells are then stimulated to become effectors, necessary to provide protective immunity.

The ability of DCs to induce adaptive immunity has boosted research on DC-vaccine strategies for bacterial, viral and parasitic pathogens and cancer immunotherapy. In fact, clinical trials are ongoing utilizing DC-mediated immunotherapy for several tumor types, including solid and hematological tumors (8). However, the clinical outcome has been inconsistent, probably associated with variable efficiency in generating DCs in vitro: autologous monocytes give origin to less-efficient DCs, and hematopoietic progenitors are isolated in very low numbers. In addition, these precursor cells are commonly compromised in cancer-bearing patients, resulting in the generation of dysfunctional DCs (8, 9). Cancer evasion mechanisms also may be underlying the lack of consistent therapeutic advantages in DC-based immunotherapies. During tumor progression cancer cells exploit several immunological processes to escape immune surveillance. These adaptations, together with cancer antigen heterogeneity, prevent the recognition of tumor antigens by the immune system and are consequently responsible for the reduced immunogenicity of tumor cells and current immunotherapies.

The generation of APCs by direct reprogramming opens new opportunities to a better understanding of DC specification and cellular identity, contributing to a more efficient control of immune responses using autologous-engineered cells.

These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

SUMMARY

The present subject matter identifies several isolated transcription factors that surprisingly reprogram or induce differentiated cell, multipotent or pluripotent stem cell into dendritic cell, in vitro, ex vivo or in vivo.

Surprisingly the induced Dendritic Cells generated by reprograming as described in the present disclosure, are intrinsically more mature than splenic DCs (natural DCs) and are less dependent on exogenous activation stimuli for antigen presentation.

DCs are professional APCs located throughout the body functioning at the interface of the innate and adaptive immune system. DCs are able to provide a crucial link between the external environment and the adaptive immune system through their ability to capture, process and present antigens to T cells, targeting them to different types of immune responses or to tolerance. Firstly, DCs have to capture antigens and process them through major histocompatibility complex (MHC) class I and MHC class II. Following their activation, DCs are able to migrate towards the local draining lymph nodes priming multiple B cell and T cell responses, a key feature of adaptive immunity. The early protective efficacy is primarily conferred by the induction of antigen-specific antibodies produced by B lymphocytes. The long-term protection against specific antigens requires the persistence of specific antibodies and the generation of immunological memory that could provide a rapid and efficient response after subsequent antigen exposure. DCs, as professional APCs, have the ability to cross-present antigens, meaning that, in addition to its classical ability to present exogenous antigens on MHC class II and endogenous antigens on MHC class I, they are also able to present exogenous antigens on MHC class I, a critical step for the generation of Cytotoxic T Lymphocyte responses (CTL).

The ontogeny and/or microenvironment in which DC are positioned may result in the expression of distinct combinations of surface receptors by DCs. For example, phenotypic criteria alone allow the classification of mouse DCs into different subpopulations.

Of these, conventional DC (cDC) in lymphoid tissues are traditionally sub-divided into cDC1 and cDC2 subpopulations. It has been argued that different DC subsets may be involved in specific recognition of certain pathogens and/or regulate different immune responses, e.g. Th1 or Th2 (immunity) or regulatory T cells (tolerance). However, the phenotype and functional behavior of DCs is also significantly conditioned by external activating stimuli, denoting significant plasticity. cDC1 and cDC2 subsets differentially prime Th1 and Th2 responses in vivo. Immune therapy for cancer relies on using DCs to prime Th1 or cytotoxic T lymphocyte responses to promote tumor clearance.

Currently, DC-based immunotherapies rely on autologous DC precursors: either monocytes, which are associated with the production of less-efficient DCs, or hematopoietic progenitors, which are isolated in very low numbers. In addition, these precursor cells are commonly compromised in cancer-bearing patients, resulting in the generation of dysfunctional DCs. In contrast, non-hematopoietic cell-types such as fibroblasts are usually not affected. Human Dermal Fibroblasts (HDFs) also exhibit other competitive advantages, namely are easily obtained from a small skin punch biopsy, are easily expanded in vitro for several passages (15-20 million cells after 4 weeks) and can be conserved frozen and used on-demand. Given the fundamental role of DCs as APCs functioning at the interface of the innate and adaptive immune system, there remains a clinical need to find alternative strategies to generate functional DCs to prime antigen-specific immune responses.

An aspect of the present disclosure relates to compositions comprising the combination of at least two isolated transcription factors encoded by a sequence 90% identical to a sequence from a list consisting of: BATF3 (SEQ. ID. 1 or SEQ. ID. 2), IRF8 (SEQ. ID. 5, SEQ. ID. 6), PU.1 (SEQ. ID. 7, SEQ. ID. 8), TCF4 (SEQ. ID. 13, SEQ. ID. 14); as a reprogramming or inducing factor of a cell selected from a list consisting of: stem cell or a differentiated cell, or mixtures thereof, into dendritic cell or antigen presenting cell in vitro, ex vivo or in vivo.

In some embodiments, polypeptide variants or family members having the same or a similar activity as the reference polypeptide encoded by the sequences provided in the sequence listing can be used in the compositions, methods, and kits described herein. Generally, variants of a particular polypeptide encoding a DC inducing factor for use in the compositions, methods, and kits described herein will have at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.

Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (over the whole the sequence) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul. 10; 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. The sequence identity values, which are indicated in the present subject-matter as a percentage were determined over the entire amino acid sequence, using BLAST with default parameters.

In an embodiment for better results, the combination of isolated transcription factor may be:

-   -   BATF3 (SEQ. ID. 1 or SEQ. ID. 2) and IRF8 (SEQ. ID. 5, SEQ. ID.         6); or BATF3 (SEQ. ID. 1, SEQ. ID. 2) and PU.1 (SEQ. ID. 7, SEQ.         ID. 8); or IRF8 (SEQ. ID. 5, SEQ. ID. 6) and PU.1 (SEQ. ID. 7,         SEQ. ID. 8); or TCF4 (SEQ. ID. 13, SEQ. ID. 14) and BATF3 (SEQ.         ID. 1, SEQ. ID. 2); or TCF4 (SEQ. ID. 13, SEQ. ID. 14) and IRF8         (SEQ. ID. 5, SEQ. ID. 6); or TCF4 (SEQ. ID. 13, SEQ. ID. 14) and         PU.1 (SEQ. ID. 7, SEQ. ID. 8); or BATF3 (SEQ. ID. 1, SEQ. ID.         2), IRF8 (SEQ. ID. 5, SEQ. ID. 6) and PU.1 (SEQ. ID. 7, SEQ. ID.         8); or TCF4 (SEQ. ID. 13, SEQ. ID. 14) BATF3 (SEQ. ID. 1, SEQ.         ID. 2) and IRF8 (SEQ. ID. 5, SEQ. ID. 6); or TCF4 (SEQ. ID. 13,         SEQ. ID. 14), IRF8 (SEQ. ID. 5, SEQ. ID. 6) and PU.1 (SEQ. ID.         7, SEQ. ID. 8); or TCF4 (SEQ. ID. 13, SEQ. ID. 14), BATF3 (SEQ.         ID. 1, SEQ. ID. 2), IRF8 (SEQ. ID. 5, SEQ. ID. 6) and PU.1 (SEQ.         ID. 7, SEQ. ID. 8).

In an embodiment, the isolated transcription factor of the present disclosure may be used in veterinary or human medicine, in particular in immunotherapy, or in neurodegenerative diseases, or in cancer or in infectious diseases.

In an embodiment for better results the cell may be selected from a list consisting of: pluripotent stem cell, multipotent stem cell, differentiated cell, tumor cell, cancer cell, and mixtures thereof. In particular mammalian cell, more in particular a mouse or a human cell.

In an embodiment for better results, the isolated transcription factor of the present disclosure may be use as a reprogramming or inducing factor of a cell selected from a list consisting of: pluripotent stem cell, or multipotent stem cell, or differentiated cell, and mixtures thereof into dendritic cell.

In an embodiment for better results, the isolated transcription factor of the present disclosure may be use a reprogramming or inducing factor of a cell selected from a list consisting of: tumor cell, cancer cell, and mixtures thereof, into antigen presenting cell.

Another aspect of the present disclosure is the use of a combination of at least two sequences at least 90% identical, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or identical, to a sequence from a list consisting of: BATF3 (SEQ. ID. 1 or SEQ. ID. 2), IRF8 (SEQ. ID. 5, SEQ. ID. 6), PU.1 (SEQ. ID. 7, SEQ. ID. 8), TCF4 (SEQ. ID. 13, SEQ. ID. 14), and mixtures thereof; as a reprogramming or inducing factor of a cell selected from a list consisting of: stem cell or a differentiated cell, or mixtures thereof, into dendritic cell or antigen presenting cell in vitro, ex vivo or in vivo.

Preferably the combination may be selected from a list consisting of: BATF3 (SEQ. ID. 1 or SEQ. ID. 2), IRF8 (SEQ. ID. 5, SEQ. ID. 6), PU.1 (SEQ. ID. 7, SEQ. ID. 8), TCF4 (SEQ. ID. 13, SEQ. ID. 14), and mixtures thereof. More preferably, the isolated transcription may include the following combination: BATF3 (SEQ. ID. 1 or SEQ. ID. 2) and IRF8 (SEQ. ID. 5, SEQ. ID. 6); or BATF3 (SEQ. ID. 1, SEQ. ID. 2) and PU.1 (SEQ. ID. 7, SEQ. ID. 8); or IRF8 (SEQ. ID. 5, SEQ. ID. 6) and PU.1 (SEQ. ID. 7, SEQ. ID. 8); or TCF4 (SEQ. ID. 13, SEQ. ID. 14) and BATF3 (SEQ. ID. 1, SEQ. ID. 2); or TCF4 (SEQ. ID. 13, SEQ. ID. 14) and IRF8 (SEQ. ID. 5, SEQ. ID. 6); or TCF4 (SEQ. ID. 13, SEQ. ID. 14) and PU.1 (SEQ. ID. 7, SEQ. ID. 8); or BATF3 (SEQ. ID. 1, SEQ. ID. 2), IRF8 (SEQ. ID. 5, SEQ. ID. 6) and PU.1 (SEQ. ID. 7, SEQ. ID. 8); or TCF4 (SEQ. ID. 13, SEQ. ID. 14), BATF3 (SEQ. ID. 1, SEQ. ID. 2) and IRF8 (SEQ. ID. 5, SEQ. ID. 6); or TCF4 (SEQ. ID. 13, SEQ. ID. 14), IRF8 (SEQ. ID. 5, SEQ. ID. 6) and PU.1 (SEQ. ID. 7, SEQ. ID. 8); or TCF4 (SEQ. ID. 13, SEQ. ID. 14), BATF3 (SEQ. ID. 1, SEQ. ID. 2), IRF8 (SEQ. ID. 5, SEQ. ID. 6) and PU.1 (SEQ. ID. 7, SEQ. ID. 8).

Another aspect of the present disclosure relates to a construct or a vector encoding at least one isolated transcription factor described in the present subject-matter.

In an embodiment for better results, the construct or the vector may be the combination of three isolated transcription factors is in the following sequential order from 5′ to 3′: PU.1 (SEQ. ID. 7, SEQ. ID. 8), IRF8 (SEQ. ID. 5, SEQ. ID. 6), BATF3 (SEQ. ID. 1, SEQ. ID. 2); or IRF8 (SEQ. ID. 5, SEQ. ID. 6), PU.1 (SEQ. ID. 7, SEQ. ID. 8), BATF3 (SEQ. ID. 1, SEQ. ID. 2).

In an embodiment, the vector is a viral vector; in particular a retrovirus, a adenovirus, a lentivirus, a herpes virus, a pox virus, or adeno-associated virus vectors.

In an embodiment for better results, the transducing step further comprises at least one vector selected from a list consisting of: a nucleic acid sequence encoding IL12; nucleic acid sequence encoding GM-CSF; nucleic acid sequence encoding IL-7; nucleic acid sequence encoding siRNA targeting IL-10 RNA, and mixtures thereof.

In an embodiment for better results the transducing of step further comprises at least one vector comprising nucleic acids encoding immunostimulatory cytokines.

Another aspect of the present disclosure relates to a method for programming or inducing a stem cell or a differentiated cell into a dendritic cell or antigen presenting cell, comprising the following step:

-   -   transducing a cell selected from a list consisting of: a stem         cell or a differentiated cell, and mixtures thereof,     -   with one or more vectors comprising at least two nucleic acid         sequence encoding a sequence at least 90% identical to a         sequence from a list consisting of: BATF3 (SEQ. ID. 1, SEQ. ID.         2), IRF8 (SEQ. ID. 5, SEQ. ID. 6), PU.1 (SEQ. ID. 7, SEQ. ID.         8), TCF4 (SEQ. ID. 13, SEQ. ID. 14), and mixtures thereof;         preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,         or identical;     -   culturing the transduced somatic cell in a cell media that         supports growth of dendritic cells or antigen presenting cells.

Preferably for better results, wherein the combination of isolated transcription factors is selected from the following encoded combinations: BATF3 (SEQ. ID. 1 or SEQ. ID. 2) and IRF8 (SEQ. ID. 5, SEQ. ID. 6); or BATF3 (SEQ. ID. 1, SEQ. ID. 2) and PU.1 (SEQ. ID. 7, SEQ. ID. 8); or IRF8 (SEQ. ID. 5, SEQ. ID. 6) and PU.1 (SEQ. ID. 7, SEQ. ID. 8); or TCF4 (SEQ. ID. 13, SEQ. ID. 14) and BATF3 (SEQ. ID. 1, SEQ. ID. 2); or TCF4 (SEQ. ID. 13, SEQ. ID. 14) and IRF8 (SEQ. ID. 5, SEQ. ID. 6); or TCF4 (SEQ. ID. 13, SEQ. ID. 14) and PU.1 (SEQ. ID. 7, SEQ. ID. 8); or BATF3 (SEQ. ID. 1, SEQ. ID. 2), IRF8 (SEQ. ID. 5, SEQ. ID. 6) and PU.1 (SEQ. ID. 7, SEQ. ID. 8); or TCF4 (SEQ. ID. 13, SEQ. ID. 14) BATF3 (SEQ. ID. 1, SEQ. ID. 2) and IRF8 (SEQ. ID. 5, SEQ. ID. 6); or TCF4 (SEQ. ID. 13, SEQ. ID. 14), IRF8 (SEQ. ID. 5, SEQ. ID. 6) and PU.1 (SEQ. ID. 7, SEQ. ID. 8); or TCF4 (SEQ. ID. 13, SEQ. ID. 14), BATF3 (SEQ. ID. 1, SEQ. ID. 2), IRF8 (SEQ. ID. 5, SEQ. ID. 6) and PU.1 (SEQ. ID. 7, SEQ. ID. 8).

In an embodiment for better results, the construct or the vector may be the combination of at least three isolated transcription factors in the following sequential order from 5′ to 3′: PU.1 (SEQ. ID. 7, SEQ. ID. 8), IRF8 (SEQ. ID. 5, SEQ. ID. 6), BATF3 (SEQ. ID. 1, SEQ. ID. 2); or IRF8 (SEQ. ID. 5, SEQ. ID. 6), PU.1 (SEQ. ID. 7, SEQ. ID. 8), BATF3 (SEQ. ID. 1, SEQ. ID. 2).

In an embodiment for better results, cells may be transduced with a plurality of isolated transcription factors and cultured during at least 2 days, preferably at least 5 days, more preferably at least 8 days, even more preferably 9 days.

In an embodiment for better results, the transducing step may further comprise at least one vector selected from a list consisting of: a nucleic acid sequence encoding IL-12; nucleic acid sequence encoding GM-CSF; nucleic acid sequence encoding IL-7; nucleic acid sequence encoding siRNA targeting IL-10 RNA, and mixtures thereof.

In an embodiment for better results, the cell may be selected from the group consisting of pluripotent stem cell, or multipotent stem cell, differentiated cell, and mixtures thereof. In particular an endoderm derived cell, a mesoderm derived cell, or an ectoderm derived cell, a multipotent stem cell including mesenchymal stem cell, a hematopoietic stem cell, intestinal stem cell, a pluripotent stem cell, a tumor or cancer cell and cell lines.

In an embodiment for better results, the cell may be a non-human cell, preferably a mouse or a human cell, more preferably cell is a human or mouse fibroblast, or a mammal umbilical cord blood stem cell.

Another aspect of the present disclosure relates to induced dendritic cell or antigen presenting cell obtained by the method described in the present disclosure.

Another aspect of the present disclosure relates to induced antigen presenting cell obtained by the method described in the present disclosure. In particular, an induced antigen presenting cell capable to present a cancer antigen, a self-antigen, an allergen, an antigen from a pathogenic and/or infectious organism.

Another aspect of the present disclosure relates to composition comprising at least one isolated transcription factor as described in the present disclosure, or an induced dendritic cell as described in the present disclosure, or an induced antigen presenting cell as described in the present disclosure, or mixtures thereof, in a therapeutically effective amount and a pharmaceutically acceptable excipient.

In a preferably embodiment, the composition may be use in veterinary or human medicine, in particular in immunotherapy, or in neurodegenerative diseases, or in cancer or in infectious diseases.

In a preferably embodiment, the composition may further comprise an anti-viral, an analgesic, an anti-inflammatory agent, a chemotherapy agent, a radiotherapy agent, an antibiotic, a diuretic, or mixtures thereof.

In a preferably embodiment, the composition may further comprise a filler, a binder, a disintegrant, or a lubricant, or mixtures thereof.

In a preferably embodiment, the composition may be use in intradermal and transdermal therapies.

In a preferably embodiment, the composition may be use as an injectable formulation, in particular an in-situ injection.

In a preferably embodiment, the composition may be use in veterinary or human medicine, in particular in immunotherapy, or in the treatment or therapy neurodegenerative diseases, or in the treatment or therapy of cancer or in the treatment or therapy of an infectious diseases.

In a preferably embodiment, the composition may be use in the treatment, therapy or diagnostic of a central and peripheral nervous system disorder.

In a preferably embodiment, the composition may be use in the treatment therapy or diagnostic of neoplasia in particular cancer, namely solid or hematological tumors.

In a preferably embodiment, the composition may be use in the treatment, diagnostic or therapy of benign tumor, malignant tumor, early cancer, basal cell carcinoma, cervical dysplasia, soft tissue sarcoma, germ cell tumor, retinoblastoma, age-related macular degeneration, Hodgkin's lymphoma, blood cancer, prostate cancer, ovarian cancer, cervix cancer, uterus cancer, vaginal cancer, breast cancer, naso-pharynx cancer, trachea cancer, larynx cancer, bronchi cancer, bronchioles cancer, lung cancer, hollow organs cancer, esophagus cancer, stomach cancer, bile duct cancer, intestine cancer, colon cancer, colorectum cancer, rectum cancer, bladder cancer, ureter cancer, kidney cancer, liver cancer, gall bladder cancer, spleen cancer, brain cancer, lymphatic system cancer, bone cancer, pancreatic cancer, leukemia, skin cancer, or myeloma.

In a preferably embodiment, the composition may be use in the treatment, therapy or diagnostic of a fungal, viral, chlamydial, bacterial, nanobacterial or parasitic infectious disease.

In a preferably embodiment, the composition may be use in therapy or diagnostic of HIV, infection with SARS coronavirus, Asian flu virus, herpes simplex, herpes zoster, hepatitis, or viral hepatitis.

In a preferably embodiment, the composition may be use in the treatment, therapy or diagnostic of an amyloid disease in particular Amyloid A (AA) amyloidosis, Alzheimer's disease, Light-Chain (AL) amyloidosis, Type-2 Diabetes, Medullary Carcinoma of the Thyroid, Parkinson's disease, Polyneuropathy, or Spongiform Encephalopathy—Creutzfeldt Jakob disease.

Another aspect of the present disclosure relates to a vaccine for cancer comprising to compositions comprising at least one isolated transcription factor as described in the present disclosure, or an induced dendritic cell as described in the present disclosure, or an induced antigen presenting cell as described in the present disclosure, or mixtures thereof.

A kit comprising at least one of the following components: a composition comprising at least one isolated transcription factor as described in the present disclosure, or an induced dendritic cell as described in the present disclosure, or a induced antigen presenting cell as described in the present disclosure, a composition as described in the present disclosure, or a vector as described in the present disclosure, or a construct as described in the present disclosure or mixtures thereof.

An aspect of the present disclosure relates to compositions, methods, and kits for dendritic cell induction or for reprogramming cells to antigen-presenting dendritic cells (DC). In some embodiments, the compositions comprise at least one DC inducing factor. Such compositions, methods and kits can be used for inducing dendritic cells in vitro, ex vivo, or in vivo, as described herein, and these induced dendritic cells (iDCs) can be used in immunotherapies.

The compositions, methods, and kits for dendritic cell induction or for reprogramming cells to dendritic cells of the present disclosure are based, in part, in the use of a novel combination of transcription factors that permit direct reprogramming of differentiated cells to the dendritic cell state. Such compositions, nucleic acid constructs, methods and kits can be used for inducing dendritic cells in vitro, ex vivo, or in vivo, as described herein, and these induced dendritic cells can be used in immunotherapies.

In an embodiment, the present disclosure relates to the regulation of the immune system, and in particular to the use of reprogrammed dendritic cells to prime immune responses to target antigens.

In an embodiment, the resulting dendritic cell is an antigen presenting cell which activates T cells against MHC class I-antigen targets. Cancer, viral, bacterial and parasitic infections are all ameliorated by the reprogrammed dendritic cells. As reprogrammed dendritic cells are capable of cross-presenting extracellular antigens via the MHC class I pathway, they are particularly suitable for generation of cytotoxic T lymphocyte responses.

In an embodiment isolated transcription factor (or exogenous transcription factor) selected from a list consisting of: BATF3 (SEQ. ID. 1, SEQ. ID. 2), IRF8 (SEQ. ID. 5, SEQ. ID. 6), PU.1 (SEQ. ID. 7, SEQ. ID. 8), TCF4 (SEQ. ID. 13, SEQ. ID. 14), and mixtures thereof, upon forced expression in fibroblasts induce activation of the Clec9a DC-specific reporter, DC morphology and a conventional DC type 1 (cDC1) transcriptional program. Induced Dendritic Cells (iDCs) express cDC1 markers, major histocompatibility complex (MHC)-I and II at the cell surface and the co-stimulatory molecules CD80, CD86 and CD40. iDCs are able to engulf particles and upon challenge with LPS or poly I:C secrete inflammatory cytokines. iDCs present antigens to CD4+ T cells and cross-present antigens to CD8+ T cells.

In an embodiment isolated transcription factor (or exogenous transcription factor) selected from a list consisting of: BATF3 (SEQ. ID. 1, SEQ. ID. 2), IRF8 (SEQ. ID. 5, SEQ. ID. 6), PU.1 (SEQ. ID. 7, SEQ. ID. 8), upon forced expression in fibroblasts induce expression of CLEC9A and HLA-DR, typical DC markers, and DC morphology. Induced Dendritic Cells (iDCs) are able to engulf particles and soluble protein. This disclosure provides powerful new treatments for cancers and cellular infections, as well as a variety of diagnostic and cell screening assays.

In an embodiment, isolated transcription factor (or exogenous transcription factor) selected from a list consisting of: BATF3 (SEQ. ID. 1, SEQ. ID. 2), IRF8 (SEQ. ID. 5, SEQ. ID. 6), PU.1 (SEQ. ID. 7, SEQ. ID. 8), upon forced expression in cancer cell lines induce expression of CLEC9A and MHC-II at the cell surface.

In an embodiment, CLEC9A is preferentially expressed on the subset cDC1 of dendritic cells. This is an important cell type because it is capable of processing antigens derived from outside the cell and presenting them to T cells via MHC class I molecules. This is in contrast to most antigen presenting cells, which present extracellularly-derived antigens via MHC class II molecules. Consequently, this mechanism of antigen presentation is sometimes referred to as “cross-presentation”. These cells therefore play an important role in the generation and stimulation of CTL responses, which are an essential part of the immune response against intracellular pathogens, e.g. viruses and cancers.

In an embodiment, immune responses stimulated via iDCs involve proliferation of T cells, which may be CTL or helper T cells. Antigen presenting cells (and in particular iDCs) can induce proliferation of both CD8+ T cells and CD4+ T cells, and may stimulate proliferation of both types of T cells in any given immune response.

In an embodiment, iDCs may be implicated in at least Th1, Th2, and Th17-type immune responses. Thus, the methods of the invention may be applied to stimulation of various types of immune response against any antigen. However, these cells are believed to be particularly important in the generation of CTL responses, so the immune response to be stimulated is preferably a CTL response. The method may comprise determining production and/or proliferation of CTLs, which are typically T cells expressing CD8 and are capable of cytotoxic activity against cells displaying their cognate antigen in the context of MHC class I molecules.

It will therefore be further understood that iDCs may be used for the prophylaxis and/or treatment of any condition in which it is desirable to induce a CTL response, such as cancer, or infection by an intracellular parasite or pathogen, such as a viral infection.

Nevertheless, if modified, iDCs can result in proliferation of helper T cells as well as, or instead of, CTLs. Thus, the method may additionally or alternatively comprise determining production and/or proliferation of helper T cells. The helper T cells may be CD4+ T cells, and may be of Th1, Th2, Th17 or Treg type.

Under certain conditions, it is believed that iDCs may be capable of stimulating regulatory T cell (Treg) proliferation. Treg cells are characterised by the expression of the Foxp3 (Forkhead box p3) transcription factor. Most Treg cells are CD4+ and CD25+, and can be regarded as a subset of helper T cells, although a small population may be CD8+. Thus, the immune response, which is to be stimulated by a method of the present disclosure, may comprise inducing proliferation of Treg cells in response to an antigen. Given that Treg cells may be capable of modulating the response of other cells of the immune system against an antigen in other ways, e.g. inhibiting or suppressing their activity, the effect on the immune system as a whole may be to modulate (e.g. suppress or inhibit) the response against that antigen. Thus, the methods of this aspect of the invention can equally be referred to as methods of modulating (e.g. inhibiting or suppressing) an immune response against an antigen. This may be particularly useful (for example) in the treatment of autoimmune disease.

iDCs will promote antigen-specific responses. The antigen may be any protein or fragment thereof against which it is desirable to raise an immune response, in particular a CTL response, but also a Th17 response or a Treg response. These may include antigens associated with, expressed by, displayed on, or secreted by cells against which it is desirable to stimulate a CTL response, including cancer cells and cells containing intracellular pathogens or parasites. For example, the antigen may be, or may comprise, an epitope peptide from a protein expressed by an intracellular pathogen or parasite (such as a viral protein) or from a protein expressed by a cancer or tumor cell. Thus, the antigen may be a tumor-specific antigen. The term “tumor-specific” antigen should not be interpreted as being restricted to antigens from solid tumors, but to encompass antigens expressed specifically by any cancerous, transformed or malignant cell.

The invention therefore provides a primed antigen presenting cell or population thereof. By “primed” is meant that the cell has been contacted with an antigen, is presenting that antigen or an epitope thereof in the context of MHC molecules, preferably MHC I molecules, and is capable of activating or stimulating T cells to proliferate and differentiate into effector cells in response thereof.

The term “antigen” is well understood in the art and includes immunogenic substances as well as antigenic epitopes. It will be appreciated that the use of any antigen is envisioned for use in the present invention and thus includes, but is not limited to, a self-antigen (whether normal or disease-related), an infectious antigen (e.g., a microbial antigen, viral antigen, etc.), or some other foreign antigen (e.g., a food component, pollen, etc.). Loading the antigen-presenting cells with an antigen can be accomplished utilizing standard methods, for example, pulsing, transducing, transfecting, and/or electrofusing. It is envisioned that the antigen can be nucleic acids (DNA or RNA), proteins, protein lysate, whole cell lysate, or antigen proteins linked to other proteins, i.e., heat shock proteins. The antigens can be derived or isolated from a pathogenic microorganism such as viruses including HIV, influenza, Herpes simplex, human papilloma virus, Hepatitis B, Hepatitis C, EBV, Cytomegalovirus (CMV) and the like. The antigen may be derived or isolated from pathogenic bacteria such as from Chlamydia, Mycobacteria, Legionella, Meningiococcus, Group A Streptococcus, Salmonella, Listeria, Haemophilus influenzae, and the like. Still further, the antigen may be derived or isolated from pathogenic yeast including Aspergillus, invasive Candida, Nocardia, Histoplasmosis, Cryptosporidia and the like. The antigen may be derived or isolated from a pathogenic protozoan and pathogenic parasites including, but not limited to Pneumocystis carinii, Trypanosoma, Leishmania, Plasmodium and Toxoplasma gondii. In certain embodiments, the antigen includes an antigen associated with a preneoplastic or hyperplastic state. Antigens may also be associated with, or causative of cancer. Such antigens are tumor specific antigen, tumor associated antigen (TAA) or tissue specific antigen, epitope thereof, and epitope agonist thereof. Such antigens include but are not limited to carcinoembryonic antigen (CEA) and epitopes thereof such as CAP-1, CAP-1-6D, MART-1, MAGE-1, MAGE-3, GAGE, GP-100, MUC-1, MUC-2, point mutated ras oncogene, normal and point mutated p53 oncogenes, PSMA, tyrosinase, TRP-1 (gp75), NY-ESO-1, TRP-2, TAG72, KSA, CA-125, PSA, HER-2/neu/c-erb/B2, BRC-I, BRC-II, bcr-abl, pax3-fkhr, ews-fli-1, modifications of TAAs and tissue specific antigen, splice variants of TAAs, epitope agonists, and the like.

The term “agent” as used herein means any compound or substance such as, but not limited to, a small molecule, nucleic acid, polypeptide, peptide, drug, ion, etc. An “agent” can be any chemical, entity or moiety, including without limitation synthetic and naturally-occurring proteinaceous and non-proteinaceous entities. In some embodiments, an agent is nucleic acid, nucleic acid analogues, proteins, antibodies, peptides, aptamers, oligomer of nucleic acids, amino acids, or carbohydrates including without limitation proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications and combinations thereof etc. In some embodiments, the nucleic acid is DNA or RNA, and nucleic acid analogues, for example can be PNA, pcPNA and LNA. A nucleic acid may be single or double stranded, and can be selected from a group comprising; nucleic acid encoding a protein of interest, oligonucleotides, PNA, etc. Such nucleic acid sequences include, for example, but not limited to, nucleic acid sequence encoding proteins that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc. A protein and/or peptide agent or fragment thereof, can be any protein of interest, for example, but not limited to; mutated proteins; therapeutic proteins; truncated proteins, wherein the protein is normally absent or expressed at lower levels in the cell. Proteins of interest can be selected from a group comprising; mutated proteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins and fragments thereof.

As used herein, the term “transcription factor” or “TF” refers to a protein that binds to specific parts of DNA using DNA binding domains and is part of the system that controls the transcription of genetic information from DNA to RNA.

The term “DC inducing factor,” as used herein, refers to a developmental potential altering factor, as that term is defined herein, such as a protein, RNA, or small molecule, the expression of which contributes to the reprogramming of a cell, e.g. a somatic cell, to the DC state. A DC inducing factor can be, for example, transcription factors that can reprogram cells to the DC state, such as PU.1, IRF8, BATF3 and TCF4, and the like, including any gene, protein, RNA or small molecule that can substitute for one or more of these factors in a method of making iDCs in vitro. In some embodiments, exogenous expression of a DC inducing factor induces endogenous expression of one or more DC inducing factors, such that exogenous expression of the one or more DC inducing factor is no longer required for stable maintenance of the cell in the iDC state.

The term “an antigen-presenting cell” (APC) as used herein, refers to a cell that displays antigen complexed with major histocompatibility complexes (MHCs) on their surfaces; this process is known as antigen presentation. T cells may recognize these complexes using their T cell receptors (TCRs). These cells process antigens and present them to T-cells.

The term “a somatic cell” used herein, refers to any biological cell forming the body of an organism; that is, in a multicellular organism, any cell other than a gamete, germ cell, gametocyte or undifferentiated stem cell.

The expression of endogenous DC inducing factors can be induced by the use of DNA targeting systems able to modulate mammalian gene expression in a cell with or without the use of chromatin modifying drugs. In some embodiments, the DNA targeting system may comprise a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) 9-based system (as described in WO2014197748 A2) that may include any modified protein, isolated polynucleotide or vector contacted with the cell and at least one guide RNA targeting a promoter region of at least one gene selected from the group consisting of PU.1, IRF8, BATF3 and TCF4. The DNA targeting system may comprise dCas9-VP64. In some embodiments, the DNA targeting system may comprise two or more transcription activator-like effector transcription factors (as described in US20140309177 A1) that bind to different target regions of at least one gene selected from the group consisting of PU.1, IRF8, BATF3 and TCF4.

In an embodiment, iDCs can be used as immunotherapy to induce specific immune responses in patients with cancer, such as melanoma, prostate cancer, glioblastoma, acute myeloid leukemia, among others.

In an embodiment, iDCs can also be used to treat infections caused by viral, bacterial and parasitic pathogens.

In an embodiment, iDCs can also be used as in vitro tools for vaccine immunogenicity testing.

The pluripotent stem cells used in the present disclosure are obtained without having to recur to a method necessarily involving the destruction of human embryos, namely with the use of induced pluripotent stem cells. Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from adult cells by cellular reprogramming.

The induced Dendritic Cells (iDCs) obtainable by this method express MHC-I and II at the cell surface and the co-stimulatory molecules CD80, CD86 and CD40.

In some embodiments, the composition may comprises the isolated transcription factor discloses in the present subject-matter, in an amount effective to improve the immunotherapy by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 95.7%, at least 98%, or at least 99% in the subject.

In some embodiments, the composition may comprise the induced dendritic cells disclosure in the present subject-matter, in an amount effective to improve the immunotherapy by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 95.7%, at least 98%, or at least 99% in the subject.

DC inducing factors of the present disclosure can be delivered to induce reprogramming in vitro, ex vivo or in vivo.

Differentiated cells of the present disclosure can be isolated from a subject in need, DC inducing factors can be introduced to induce reprogramming into iDCs. Generated iDCs can be infused back to the patient.

Alternatively, DC inducing factors can be delivered to induce reprogramming in vivo of, for example, cancer cells into iDC with ability to present cancer antigens.

Preferred routes of administration include but are not limited to oral, parenteral, intramuscular, intravenous, in situ injection, intranasal, sublingual, intratracheal, and inhalation.

In some embodiments, the dose or dosage form is administered to the subject once a day, twice a day, or three times a day. In other embodiments, the dose is administered to the subject once a week, once a month, once every two months, four times a year, three times a year, twice a year, or once a year.

The embodiments of the invention provide multiple applications, including kits for research use and methods for generation of cells useful for conducting small molecule screens for immune disorders. In addition, the invention provides commercially and medically useful methods to produce autologous dendritic cells and give them back to a patient in need.

For example, the methods described herein can be used to produce dendritic cells to treat diseases including hyperproliferative diseases, which can also be further defined as cancer. In still further embodiments, the cancer is melanoma, non-small cell lung, small-cell lung, lung, hepatocarcinoma, leukemia, retinoblastoma, astrocytoma, glioblastoma, gum, tongue, neuroblastoma, head, neck, breast, pancreatic, prostate, colorectal, Esophageal, Non-Hodgkin lymphoma, uterine, liver, thyroid, renal, skin, bone, testicular, ovarian, mesothelioma, cervical, gastrointestinal, lymphoma, brain, colon, sarcoma or bladder. The cancer may include a tumor comprised of tumor cells. For example, tumor cells may include, but are not limited to melanoma cell, a bladder cancer cell, a breast cancer cell, a lung cancer cell, a colon cancer cell, a prostate cancer cell, a liver cancer cell, a pancreatic cancer cell, a stomach cancer cell, a testicular cancer cell, a brain cancer cell, an ovarian cancer cell, a lymphatic cancer cell, a skin cancer cell, a brain cancer cell, a bone cancer cell, or a soft tissue cancer cell. In other embodiments, the hyperproliferative disease is rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis, pre-neoplastic lesions (such as adenomatous hyperplasia and prostatic intraepithelial neoplasia), carcinoma in situ, oral hairy leukoplakia, or psoriasis.

Accordingly, provided herein, in an embodiment are dendritic cell (DC) inducing composition comprising one or more expression vectors encoding at least two, three, four, or more DC inducing factors selected from: BATF3 (SEQ. ID. 1, SEQ. ID. 2), IRF8 (SEQ. ID. 5, SEQ. ID. 6), PU.1 (SEQ. ID. 7, SEQ. ID. 8), TCF4 (SEQ. ID. 13, SEQ. ID. 14), or mixtures thereof. In a particular embodiment, the addition of increases the efficiency in at least 8%.

In some embodiments of these aspects and all such aspects described herein, the one or more expression vectors are retroviral vectors.

In some embodiments of these aspects and all such aspects described herein, the one or more expression vectors are lentiviral vectors. In some embodiments, the lentiviral vectors are inducible lentiviral vectors.

Also provided herein, in some aspects, are dendritic cell (DC) inducing compositions comprising modified mRNA sequences encoding at least two, three, four, DC inducing factors selected from BATF3 (SEQ. ID. 1, SEQ. ID. 2), IRF8 (SEQ. ID. 5, SEQ. ID. 6), PU.1 (SEQ. ID. 7, SEQ. ID. 8), TCF4 (SEQ. ID. 13, SEQ. ID. 14), or mixtures thereof, wherein each cytosine of each said modified mRNA sequence is a modified cytosine, each uracil of each said modified mRNA sequence is a modified uracil, or a combination thereof.

Provided herein, in some aspects, are dendritic cell (DC) inducing compositions comprising at least two sequences selected from TCF4 (SEQ. ID. 13, SEQ. ID. 14), BATF3 (SEQ. ID. 1, SEQ. ID. 2), IRF8 (SEQ. ID. 5, SEQ. ID. 6), PU.1 (SEQ. ID. 7, SEQ. ID. 8), or mixtures thereof, wherein each cytosine of each said modified mRNA sequence is a modified cytosine, each uracil of each said modified mRNA sequence is a modified uracil, or a combination thereof.

In some embodiments of these aspects and all such aspects described herein, the modified cytosine is 5-methylcytosine and the modified uracil is pseudouracil.

Also provided herein in some aspects, are methods for preparing an induced dendritic cell (iDC) from a somatic cell comprising:

-   -   transducing the somatic cell with one or more vectors comprising         a nucleic acid sequence encoding PU.1 (SEQ. ID. 7, SEQ. ID. 8),         a nucleic acid sequence encoding IRF8 (SEQ. ID. 5, SEQ. ID. 6);         a nucleic acid sequence encoding BATF3 (SEQ. ID. 1, SEQ. ID. 2);         wherein each said nucleic acid sequence is operably linked to a         promoter; and     -   culturing the transduced somatic cell in a cell media that         supports growth of dendritic cells, thereby preparing an iDC.

In some embodiments of these aspects and all such aspects described herein, the transducing step further comprises one or more vectors comprising one or more of: a nucleic acid sequence encoding TCF4 (SEQ. ID. 13, SEQ. ID. 14); a nucleic acid sequence encoding IL12; nucleic acid sequence encoding GM-CSF; nucleic acid sequence encoding IL-7; nucleic acid sequence encoding siRNA targeting IL-10 RNA.

In some embodiments of these aspects and all such aspects described herein, the transducing step further comprises one or more vectors comprising nucleic acids encoding immunostimulatory cytokines. Preferably, the cytokine is one of the interleukins (e.g., IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-18, IL-19, IL-20), interferons (e.g., IFN-α, IFN-3, IFN-γ), tumor necrosis factor (TNF), transforming growth factor-β (TGF-β), granulocyte colony stimulating factors (G-CSF), macrophage colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), Flt-3 ligand or kit ligand. The amino acid sequences of these cytokines are well known in the art. In the case of heterodimeric immunostimulatory cytokines (e.g., IL-12), induced dendritic cells (iDCs) shall be genetically modified to express both subunits of the cytokine molecule.

The additional vectors may also comprise nucleic acids encoding variants of these cytokines. For example, for those cytokines having both pro-forms and mature forms (e.g., before and after cleavage of a signal peptide, or before and after limited proteolysis to yield an active fragment), the APCs of the invention may be genetically modified to express either the pro- or mature form. Other variants, such as fusion proteins between an active fragment of a cytokine and a heterologous sequence (e.g., a heterologous signal peptide), may also be employed. Species variants may also be employed to the extent that they retain activity in a human subject. Thus, for example, human APCs may be genetically modified to express a murine, bovine, equine, ovine, feline, canine, non-human primate or other mammalian variant of a human cytokine if these species variants retain activity substantially similar to their human homologues.

It may be desirable also to administer further immunostimulatory agents in order to achieve maximal CTL stimulation and proliferation, and/or stimulation and proliferation of other T cell types. These may include agents capable of activating dendritic cells and stimulating their ability to promote T cell activation. Such an agent may be referred to as an adjuvant. The adjuvant may comprise an agonist for CD40 (such as soluble CD40 ligand, or an agonist antibody specific for CD40), an agonist of CD28, CD27 or OX40 (e.g. an agonist antibody specific for one of those molecules), a CTLA-4 antagonist (e.g. a blocking antibody specific for CTLA-4), and/or a Toll-like receptor (TLR) agonist, and/or any other agent capable of inducing dendritic cell activation. A TLR agonist is a substance that activates a Toll-like receptor. Preferably, the TLR agonist is an activator of TLR3, TLR4, TLR5, TLR7 or TLR9. A suitable TLR agonist is MPL (monophosphoryl lipid A), which binds TLR4. Other TLR agonists which may be used are LTA (lipoteichoic acid, which binds TLR2; Poly I:C (polyinosine-polycytidylic acid), which binds TLR3; flagellin, which binds TLR5; imiquimod or polyU RNA (1-(2-methylpropyl)-1H-imidazo(4,5-c)quinolin-4-amine), which binds TLR7 and CpG (DNA CpG motifs), which binds TLR9; or any other component which binds to and activates a TLR. For more details, see Reis e Sousa, Toll-like receptors and dendritic cells. Seminars in Immunology 16:27, 2004. Adjuvants which may not work via TLRs include 5′ triphosphate RNA, poly I:C, and β-glucans such as curdlan (β-1,3-glucan).

In some embodiments of these aspects and all such aspects described herein, the culturing step further comprises the use of cell media that supports growth of dendritic cells or antigen presenting cells supplemented with at least one immunostimulatory recombinant cytokine selected from the group consisting of interleukins (e.g., IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-18, IL-19, IL-20), interferons (e.g., IFN-α, IFN-(3, IFN-γ), tumor necrosis factor (TNF), transforming growth factor-β (TGF-β), granulocyte colony stimulating factors (G-CSF), macrophage colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), Flt-3 ligand or kit ligand. In the case of heterodimeric immunostimulatory cytokines (e.g., IL-12), induced dendritic cells (iDCs) shall be cultured with both subunits of the cytokine molecule. Other pro-inflammatory cytokines may also be used as adjuvants.

In some embodiments of these aspects and all such aspects described herein, the somatic cell is a fibroblast cell.

In some embodiments of these aspects and all such aspects described herein, the somatic cell is a hematopoietic lineage cell.

In some embodiments of these aspects and all such aspects described herein, the hematopoietic lineage cell is selected from promyelocytes, neutrophils, eosinophils, basophils, reticulocytes, erythrocytes, mast cells, osteoclasts, megakaryoblasts, platelet producing megakaryocytes, platelets, monocytes, macrophages, lymphocytes, NK cells, NKT cells, innate lymphocytes, multipotent hematopoietic stem and progenitor cells, oligopotent hematopoietic progenitor cells, lineage restricted hematopoietic progenitors.

In some embodiments of these aspects and all such aspects described herein, the hematopoietic lineage cell is selected from a multi-potent progenitor cell (MPP), common myeloid progenitor cell (CMP), granulocyte-monocyte progenitor cells (GMP), common lymphoid progenitor cell (CLP), and pre-megakaryocyte-erythrocyte progenitor cell.

In some embodiments of these aspects and all such aspects described herein, the hematopoietic lineage cell is selected from a megakaryocyte-erythrocyte progenitor cell (MEP), a ProB cell, a PreB cell, a PreProB cell, a ProT cell, a double-negative T cell, a pro-NK cell, a pre-dendritic cell (pre-DC), pre-granulocyte/macrophage cell, a granulocyte/macrophage progenitor (GMP) cell, and a pro-mast cell (ProMC).

Also provided herein, in some aspects, are kits for making induced dendritic cells (iDC), the kits comprising any of the DC inducing compositions comprising one or more expression vector components described herein.

Provided herein, in some aspects, are kits for making induced dendritic cells (iDC), the kits comprising any of the DC inducing compositions comprising modified mRNA sequence components described herein.

In some embodiments of these aspects and all such aspects described herein, the one or more expression vectors are lentiviral vectors. In some embodiments, the lentiviral vectors are inducible lentiviral vectors. In some embodiments, the lentiviral vectors are polycistronic inducible lentiviral vectors. In some embodiments, the polycistronic inducible lentiviral vectors express three or more nucleic acid sequences. In some embodiments, each of the nucleic acid sequences of the polycistronic inducible lentiviral vectors are separated by 2A peptide sequences.

The use of polycistronic viral expression systems can increase the in vivo reprogramming efficiency of somatic cells to iDCs. Accordingly, in some embodiments of the aspects described herein, a polycistronic lentiviral vector is used. In such embodiments, sequences encoding two or more of the DC inducing factors described herein, are expressed from a single promoter, as a polycistronic transcript. 2A peptide strategy can be used to make polycistronic vectors (see, e.g., Expert Opin Biol Ther. 2005 May; 5(5):627-38). Polycistronic expression vector systems can also use internal ribosome entry sites (IRES) elements to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5′-methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, thus creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message. See, for example, U.S. Pat. Nos. 4,980,285; 5,925,565; 5,631,150; 5,707,828; 5,759,828; 5,888,783; 5,919,670; and 5,935,819; and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989).

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of invention.

FIG. 1. Schematic representation of hematopoietic differentiation. Whereas hematopoietic differentiation normally proceeds from hematopoietic stem cells (HSCs) through progressively more-restricted progenitors into differentiated blood effector cells, such as dendritic cells (DCs) (highlighted with dashed-line box), the results described herein aim to utilize DC-enriched transcription factors to reprogram somatic differentiated cells from other lineages into the dendritic cell fate with antigen-presenting capacity. Multi-potent progenitor cells (MPPs), common myeloid progenitor cells (CMPs), common lymphoid progenitor cells (CLPs), granulocyte-monocyte progenitor cells (GMPs), pre-megakaryocyte-erythrocyte (pre-MEG/E) progenitor cell, megakaryocyte-erythrocyte progenitor cells (MEP), colony forming unit-erythroid (CFU-E), megakaryocytic progenitor MkP, pro-mast cells (ProMCs), Monocyte DC progenitors (MDP), Common Dendritic Cell Precursors (CDP), pre-dendritic cell (pre-DC), double negative T lineage precursors (DN1, DN2).

FIG. 2. Generating Antigen Presenting Cells by Direct Cellular Reprogramming. Observation of the effect of the TF combination disclosure in the present subject-matter for the induction of dendritic cells (iDCs) from mouse and human fibroblasts. Induced DCs can be applied to generate a personalized immunotherapy after loading with cell extracts or defined antigens (Primed-iDC). DCs are specialized in antigen presentation to Macrophages (Mø), T, B and NK cells. Induced DCs stimulate antigen-specific immune responses against cancer, viral, parasitic or bacterial infections.

FIG. 3. In situ or ex vivo direct reprogramming of cancer cells to stimulate antigen-specific immune responses. Effect of TF combination (PIB) for the induction of DC fate and antigen presenting capacity, when this cocktail is introduced directly into cancer cells in vivo or in situ or, ex vivo or in vitro. This strategy enables tumor cells to present their specific antigens (Tumor-APC) to CD4+ and CD8+ T-cells, triggering a targeted immune response against the tumor.

FIG. 4. 18 TF candidates for the direct reprogramming of DCs. (A) Heat map showing gene expression of the 18 candidate factors across multiple mouse tissues (GeneAtlas MOE430). The majority of the 18 factors are specifically enriched in DCs (black box) but not in other tissues (right). (B) Heat maps showing increased gene expression of the 18 factors in mouse DCs when compared with macrophages (Mcp) derived from bone marrow cultures (left panel, GSE62361). Heat maps displaying gene expression of the 18 TFs in common dendritic cell precursors (CDP), Pre-conventional DCs (Pre-cDC1 and Pre-cDC2) and conventional DCs (cDC1 and cDC2) (right panel, GSE66565). Gene expression data were analyzed by Cluster 3.0 and displayed by Treeview. Red indicates increased expression, whereas blue indicates decreased expression over the mean. (C) Gene ontology biological process (left) and mouse loss-of-function mutant phenotype (right) enrichment analysis was performed for the candidate 18 TFs using Enrichr (http://amp.pharm.mssm.edu/Enrichr/). Lists show the most enriched terms (top 19) and left columns show respective p-values. Top enriched biological processes enriched are leucocyte differentiation (p=2.51E-12), leucocyte activation (p=1.02E-11), DC differentiation (p=9.58E-12) and DC activation (p=6.31E-11), whilst mutant phenotypes include abnormal adaptive immunity (p=1.19E-04) and abnormal antigen presentation (p=1.25E-03).

FIG. 5. Expression of Clec9a is specifically restricted to the conventional DC-lineage. (A) Clec9a-Cre X R26-stop-Tomato double transgenic mouse enables identification of conventional DCs and their committed precursors (CDP, Common Dendritic Cell Precursors), but not other leukocytes, due to restricted tdTomato expression. (B) Expression profile of Clec9a in DCs and several hematopoietic cell lineages obtained from data available in Immunological Genome Project (www.immgen.org). (C) Gene expression of the Clec9a gene in Monocyte DC progenitors (MDPs) and DC-committed precursors (CDPs and pre-DCs) at the single cell level (GSE60783).

FIG. 6. Clec9a is highly expressed in mature cDC1s. (A) Gene expression of Clec9a in DC precursors (CDPs, pre-DC1 and pre-DC2) and mature cells (cDC1 and cDC2) (GSE60782). (B) Confirmation of Clec9a-tdTomato on splenic cDC1 (MHC-II+ CD11c+ CD8+ cells) isolated from double transgenic C9a-tdT animals. CD8+ T-cells that do not express Clec9a were included as control.

FIG. 7. Isolation and purification of Clec9a reporter MEFs to screen candidate TFs. (A) Double transgenic (Clec9a-Cre X R26-stop-Tomato) pregnant females were used to isolate MEFs at embryonic day E13.5. After removal of the head, fetal liver and internal organs, MEFs were cultured until confluency. MEFs were sorted to remove residual CD45+ and tdTomato+ cells that could represent cells with hematopoietic potential. (B) Gating strategy to remove residual CD45+ and tdTomato+ cells. Double negative MEFs, approximately 97% of the population, were sorted. (C) Purity confirmation of the sorted population.

FIG. 8. Experimental design to screen candidate TFs' ability to activate Clec9a reporter MEFs. Purified MEFs were transduced with different pools of inducible lentiviral vectors encoding DC-specific TFs. MEFs were cultured in the presence of Dox to induce expression of the TFs and monitored from day 1 to 15 for tdTomato expression. Activation of Clec9a promoter induces expression of Cre recombinase, which mediates excision of the Stop codon and consequent expression of tdTomato.

FIG. 9. Combinations of candidate DC-inducing TFs induce activation of the Clec9a-tdTomato reporter. (A) MEFs were transduced with M2rtTA (as control), all 18 candidate TFs and pools of 3-4 TFs and analyzed by fluorescent microscopy and flow cytometry 5 days after addition of Dox. (B) Quantification of tdT+ cells after transduction with M2rtTA, all 18 TFs or smaller pools at day 8. Mean±SD, n=2. (C) Quantification of tdT+ cells after removal of individual TFs from the pool of 4 TFs or their individual expression at day 8. Mean±SD, n=2.

FIG. 10. Minimal transcription factor network activates Clec9a reporter and induce DC morphology in mouse fibroblasts. (A) MEFs were transduced with M2rtTA (as control) or PU.1, IRF8 and BATF3 (PIB—mixture of the 3 TF) and analysed by fluorescent microscopy and flow cytometry 5 days after addition of Dox. (B) Kinetics of Clec9a-tdTomato reporter activation analysed by flow cytometry. (C) Quantification of tdTomato+ cells after removal of individual TFs from the pool of PIB or their individual expression at day 5 after addition of Dox. (D) Flow cytometry histograms showing size (FSC) and complexity (SSC) in PIB transduced cells (gated in tdTomato+ or total population) and M2rtTA transduced cells. (E) Morphology of tdTomato+ cells at day 5 after the addition of Dox. (F) Immunofluorescence for F-actin at day 8 after addition of Dox.

FIG. 11. Kinetics of Clec9a reporter activation analyzed by time-lapse microscopy from day 0 to day 7. Scale bars represent 200 μm.

FIG. 12. Minimal transcription factor network induces expression of the pan-hematopoietic marker CD45. Flow cytometry analysis at day 8 for expression of CD45 and tdTomato in PIB-transduced MEFs.

FIG. 13. TCF4 increases the efficiency of Clec9a reporter activation. (A) MEFs were transduced with PU.1, IRF8 and BATF3, PIB (black bar) or PIB combined with individual TFs from the 18 candidates (grey bars). tdTomato+ cells were quantified at day 8. M2rtTA transduction was included as control. Mean±SD, n=2-6. (B) MEFs were transduced with PIB (black bar) or PIB combined with individual hematopoietic TFs (grey bars). tdTomato+ cells were quantified at day 8. Mean±SD, n=2-6.

FIG. 14. Optimal culture conditions to induce the activation of the Clec9a reporter. (A) Quantification of tdTomato+ cells in MEFs transduced with PIB (PU.1, IRF8 and BATF3) and cultured in different conditions at day 10 after addition of Dox. (B) Absolute numbers of tdTomato+ cells in MEFs transduced with PIB (black bar) and co-cultured with OP9 and OP9-DL1 cells at day 10 after addition of Dox. OP9 and OP9-DL1 cultures were included as controls.

FIG. 15. Expression profiles of PU.1, IRF8, BATF3 and TCF4 at the single cell level. Gene expression of PU.1, IRF8, BATF3 and TCF4 in single monocyte-dendritic cells precursors (MDPs) and restricted DC precursor cells (CDPs, and pre-DCs) (GSE60783). Gene expression level is shown in reads per kilobase of exon model per million mapped reads (RPKM) values.

FIG. 16. Analysis of PU.1, IRF8, BATF3 factors expression in mouse cells/tissues. (A) Combined gene expression levels of Pu.1+Ir8+Batf3 across 96 tissues and cell-types. Gene expression data from different mouse tissues and cell-types, were obtained from the GeneAtlas MOE430 database. The “GPSforGenes” program was written and used to classify the tissues where combinations of TFs were most enriched (best fit=1). (B) Gene expression of Pu.1, Irf8 and Batf3 in DC precursors (CDPs, pre-DC1 and pre-DC2) and mature cells (cDC1 and cDC2) (GSE60782). Gene expression level is shown in relative gene expression. (C) Heat map showing increased expression of Clec9a, PU.1, IRF8 and BATF3 in CD103+ DCs (highlighted in red) belonging to cDC1 subset when compared to other DC subsets and several hematopoietic cell lineages available in the Immunological Genome Project (www.immgen.org). Gene expression data were analyzed by Cluster 3.0 and displayed by Treeview. Red indicates increased expression, whereas blue indicates decreased expression over the mean.

FIG. 17. Induced DCs express cDC1 markers at cell surface. (A) Flow cytometry analysis of surface phenotype of MEFs transduced with PIB 8 days after the addition of Dox. Quantification of CD103, CD8a, CD4, C11b and B220 expression in tdT+ and tdT-populations. (B) Representative flow cytometry plots.

FIG. 18. Induced DCs express antigen-presenting machinery at the cell surface (A) Flow cytometry analysis of MHC-II expression in MEFs transduced with M2rtTA (as control) and PIB (PU.1, IRF8 and BATF3) 7 days after addition of Dox. TdTomato+ and tdTomato-populations are shown. (B) Kinetics of MHC-II surface expression in M2rtTA and PIB transduced cells. MEFs were analysed by flow cytometry from day 1 to 15 after addition of Dox. (C) Quantification of the percentage of cells expressing MHC-II in high and low levels in bulk cultures after removal of individual TF from the pool of PIB, at day 5 after addition of Dox. (D) Quantification of MHC-II+ cells at day 5 within tdTomato+ population after transduction with 4TFs or upon their individual removal.

FIG. 19. Induced DCs express MHC-I at cell surface. Flow cytometry analysis of MHC-I expression in MEFs transduced with M2rtTA and PIB at day 7 after the addition of Dox.

FIG. 20. Induced DCs express co-stimulatory molecules at cell surface. Flow cytometry analysis of co-stimulatory molecules (CD80 and CD86) at cell surface of tdTomato+ population in PIB (PU.1, IRF8 and BATF3) transduced MEFs 5 days after addition of Dox. MHC-II+ and MHC-II-populations are shown.

FIG. 21. Induced DCs up-regulate CD40 expression upon LPS stimuli. (A) Flow cytometry analysis of CD40 expression in tdTomato- and tdTomato+ population in PIB-transduced MEFs at day 8. (B) Histograms show expression of CD40 with or without overnight LPS stimulation at day 13.

FIG. 22. PIB factors induce global dendritic cell gene expression program in fibroblasts. (A) Clec9a reporter MEFs were transduced with Pu.1 (P), Irf8 (I) and Batf3 (B) to generate iDCs. Transduced cells were sorted by FACS and sampled using full-transcript single-cell RNA-seq using Fluidigm Cl system, at day 3 (d3, 20 Clec9a-tdTomato+ cells), day 7 (d7, 40 Clec9a-tdTomato+ cells) and day 9 (d9, 36 Clec9a-tdTomato+ MHC-II+ cells). Non-transduced MEFs at day 0 (30 cells) and splenic DCs (sDCs, 66 CD11c+ MHC-II+CD8a+ cells) isolated from C57131/6 animals were used as controls. (B) t-distributed stochastic linear embedding (tSNE) analysis of genome-wide transcriptomes showing clustering of 163 single cells. Each dot represents an individual cell. The number of cells from each sample group is depicted inside brackets. (C) Complete•linkage hierarchical clustering of the consensus matrix obtained by the SC3 clustering algorithm. (D) Heatmap showing expression of the 6525 most variable genes across the 5 different biological sample groups (columns, MEFs, d3, d7, d9 and sDCs). 4 clusters of genes are shown: Cluster I (3014 genes), II (530 genes), III (347 genes) and IV (2634 genes). Color scheme is based on z-score distribution, from −2 (blue) to 2 (red). Examples of genes from each cluster are shown (right panel). (E) Expression levels of fibroblast genes are shown as Census counts median values±95% confidence interval. (F) Expression levels of genes in Cluster II (Eea1, Aldh1a2, Ifit3), Cluster III (H2-Pb, Ctsc, Cd74) and Cluster IV (Clec9a, Cd45, Cd11c, TIr3, Ccr2, Nlrc5), presented as violin plots (height, gene expression; width, abundance of cells expressing the gene). Log values of Census counts are shown, horizontal lines corresponding to median values.

FIG. 23. PIB factors induce expression of DC transcriptional regulators, including endogenous Pu.1, Irf8 and Batf3. (A) Violin plots showing the expression levels of the DC transcriptional regulators Zbtb46, Bcl11a, Stat2, Irf7, Stat6 and Stat1. (B) Expression levels of Pu.1, Irf8 and Batf3 genes are shown as Log counts presented as box plot with whiskers extending to ±1.5× interquartile range. Total (left panel) and endogenous transcript (right panel) levels are displayed.

FIG. 24. Pathway enrichment for step-wise transitions during iDC reprogramming. (A) Gene set enrichment analysis (GSEA) for step-wise iDC reprogramming was performed using annotated gene sets from NetPath-annotated signaling pathways. Day 3 refers to the pathways upregulated at day 3 versus MEFs, day 7 refers to the pathways upregulated at day 7 versus day 3 and day 9 refers to the upregulated pathways at day 9 versus day 7. Datasets were ordered according to the normalized enrichment score (NES) and the False Discovery Rate (FDR) q-value is shown. The bottom panel shows the enrichment plots for the IL-4 (day 3) and Oncostatin M (day 9) gene sets.

FIG. 25. Transcriptional networks for step-wise transitions during iDC reprogramming. (A) Transcription factor covariance networks during iDC reprogramming for each step-wise transition. Shown are transcriptional regulators with more than five edges, with each edge reflecting a correlation >0.35 between connected transcriptional regulators. The transcription factors PU.1, IRF8 and BATF3 are highlighted in red. (B) Heat maps showing expression of transcriptional regulators shown in panel A in DC precursors (CDPs and pre-DC1) and mature cells (cDC1) from bone marrow (GSE60782). Gene expression data were analyzed by Cluster 3.0 and displayed by Treeview. Red indicates increased expression, whereas blue indicates decreased expression over the mean. (C) PIB-transduced MEFs at day 3, 5, 7, 10 and 25 after addition of Dox were assayed for hematopoietic colony formation (mean±SD, n=2). Sorted sDC1s and unsorted splenocytes and bone marrow cells were included as controls.

FIG. 26. PIB factors induce transcriptional reprogramming towards cDC1 expression program. cDC1 and cDC2 gene expression signatures were generated by analyzing the datasets from Schlitzer et al. (11) (GSE60783). Cumulative median expression levels of cDC1 and cDC2 gene signatures during reprogramming.

FIG. 27. Reconstruction of single cell reprogramming trajectory. (A) Genome wide transcriptomes of single cells were ordered with TSCAN software (Pseudo-time). Ordering of nontransduced MEFs, induced DCs (iDCs) Clec9a-tdTomato+ at day 3 (d3), day 7 (d7), Clec9a-tdTomato+ MHC-11+ day 9 (d9) and splenic DCs (sDCs, CD11c+ MHC-II+ CD8a+) are shown. Each dot represents an individual cell. (B) Cell expression profiles in a two-dimensional independent component space according to predicted trajectory. Solid black line shows pseudo-time ordering constructed by Monocle2. Each dot represents an individual cell, colored according to biological sample groups (left panel) or cell state (right panel). The number of cells from each sample group assigned to each cell state is depicted inside brackets.

FIG. 28. Reconstruction of single cell reprogramming trajectory highlights different maturation states of iDCs. (A) Five kinetic clusters of branch-dependent genes identified by BEAM. (B) Gene set enrichment analysis (GSEA) between cell state 2 and cell state 3 was performed using gene sets present in the Immunologic signatures collection (4,872 gene sets, FDR<0.02 or maximum of 200 genes per gene set). Gene sets were ordered according to the normalized enrichment score (NES) and the False Discovery Rate (FDR) q-value is shown. Black lines represent DC gene sets. The right panel shows enrichment plots for Mature Stimulatory DC, IFNγ stimulated DC and IFNα stimulated DC gene sets (all enriched in State 3).

FIG. 29. PIB factors induce high levels of expression of genes associated with DC maturation. (A) GSEA for day 9 iDCs and sDC1s showing the enrichment for 2 MSigDB gene sets (left). Violin plots (right) show expression distribution of day 9 enriched genes.

FIG. 30. PIB factors induce high levels of expression of genes associated with DC maturation. (A) Expression levels of Ciita, H2-Aa, H2-Ab1 and H2-Eb1 genes in single cells from State 1, 2 and 3 ordered with Monocle2 (Pseudo-time). Each dot represents relative expression values for individual cells. Lines represent branch kinetics curves for State 2 (solid line) and State 3 (dashed line). (B) Violin plots showing the expression levels of Ciita, Acp5, Tnfrsf1a, Tapbp, Inpp5d, Traf6 and Itga4 genes. Log values of Census counts are shown, horizontal lines corresponding to median values.

FIG. 31. Induced DCs secrete inflammatory cytokines upon TLR stimuli. (A) Secretion of cytokines by MEFs transduced with PIB (PU.1, IRF8 and BATF3) or PIBT (PU.1, IRF8, BATF3 and TCF4) with or without TLR4 (LPS) or TLR3 (PolyI:C) stimuli overnight. Supernatants of MEFs transduced with PIB or PIBT factors were collected at day 10 after addition of Dox and analysed for cytokine concentration using BD Cytometric Bead Array Mouse Inflammation Kit. Cytokine levels for untreated, 100 ng/mL LPS or 25 μg/mL of PolyI:C-treated cells overnight are shown; black or grey bars represent PIB or PIBT-transduced MEFs, respectively.

FIG. 32. Induced DCs are able to engulf small particles. MEFs transduced with PIB (PU.1, IRF8 and BATF3) were incubated overnight with FITC-labelled latex beads (1 μm) and analysed by fluorescent microscopy at day 7 after addition of Dox.

FIG. 33. Induced DCs are able to engulf proteins and dead cells. (A) PIB-transduced MEFs were FACS sorted and tdTomato- and tdTomato+(iDCs) populations were incubated with AlexaFluor647-labelled Ovalbumin (OVA-Alexa647) at 37° C. at day 11 and analysed by flow cytometry. Controls were kept on ice (4° C.). (B) Sorted tdTomato- and tdTomato+(iDCs) populations at day 11 were incubated overnight with dead cells labeled with CellVue Claret Far Red membrane staining and analyzed by flow cytometry. (C, D) iDCs at day 11 were incubated overnight with dead cells labelled with DAPI and analysed by fluorescent or (D) time-lapse microscopy.

FIG. 34. Induced DCs express genes involved in TLR signalling and endocytic pathway. Violin plots for genes regulating (A) TLR signalling and (B) incorporation of antigens.

FIG. 35. Induced DCs capture and present antigens to CD4+ T cells. (A) Schematic representation of antigen presenting assay. iDCs at day 8 after addition of Dox were co-cultured with OT-II CD4+ T cells isolated from OT-II Rag2KO mice and labelled with CFSE in the presence of Ovalbumin (OVA) or OVA peptide 323-339. After 7 days, activation of CD4+ T cells was evaluated by CFSE dilution and expression of T cell activation marker CD44. (B) Flow cytometry plots of CFSE-labelled CD4+ T cells co-cultured with MEFs transduced with PIB or PIB plus TCF4, in the presence of OVA, stimulated or not with LPS. CD4+ T cells co-cultured with splenic MHC-II+ CD11c+ DCs were included as controls. Grey lines correspond to untouched CD4+ T cells. (C) Flow cytometry plots showing CD44 expression of CFSE-labelled CD4+ T cells co-cultured with MEFs transduced with PIB, in the presence of LPS and OVA or OVA peptide.

FIG. 36. Induced DCs capture and present antigens to CD4+ T cells. Quantification of the percentage of CFSElow CD4+ T-cells co-cultured with MEFs transduced with PIB (iDCs), in the presence of LPS and OVA or OVA peptide. Splenic DCs were included as control.

FIG. 37. Induced DCs efficiently export endocytic cargo into the cytoplasm and express cross-presentation genes. (A) iDCs at day 16 were loaded with a FRET-sensitive cytosolic substrate of β-lactamase, CCF4, followed by incubation with β-lactamase. Kinetics of β-lactamase's export to cytosol was measured as CCF4 cleavage by flow cytometry. (B) Violin plots for genes regulating cross-presentation.

FIG. 38. Induced DCs capture and cross-present exogenous antigens to CD8+ T cells. (A) iDCs at day 16 were co-incubated with B3Z T-cell hybridomas for 16 h and increasing concentrations of soluble OVA protein in the absence (left panel) or presence of LPS or poly-I:C (PIC) stimulation (right panel). T-cell activation was measured as up-regulation of β-galactosidase expression in B3Zs (driven by the IL-2 promoter) and quantified using a colorimetric substrate, CPRG. (B) Schematic representation of cross-presentation assay (left panel). iDCs at day 8 after addition of Dox were co-cultured with OT-1 CD8+ T cells isolated from OT-1 Rag2KO mice and labelled with CFSE in the presence of Ovalbumin (OVA) or OVA 257-264 peptide. After 4 days, activation of CD4+ T cells was evaluated by CFSE dilution and expression of the T cell activation marker CD44. Flow cytometry plots showing CD44 expression of CFSE-labelled CD8+ T cells co-cultured with MEFs transduced with PIB or PIB plus TCF4, in the presence of OVA. CD8+ T cells co-cultured with splenic MHC-II+ CD11c+ DCs were included as controls (middle panel), and respective quantification (right panel).

FIG. 39. PU.1, IRF8 and BATF3 induce DC-like morphology in human fibroblasts. (A) Human Dermal Fibroblasts (HDFs) were transduced with PIB (PU.1, IRF8, BATF3) and cultured in the presence of Dox. (B) Bright field images of HDFs transduced with PIB at day 3 after addition of Dox. White arrowheads mark cells with typical DC-like morphology. M2rtTA transduced HDFs are shown as control. (C) Higher magnification of bright field images of PIB-transduced HDFs with DC-like morphology.

FIG. 40. PU.1, IRF8 and BATF3 induce expression of HLA-DR and CLEC9A in human fibroblasts. Flow cytometry analysis of HLA-DR and CLEC9A expression in PIB-transduced human fibroblasts at day 9 after addition of Dox.

FIG. 41. PU.1, IRF8 and BATF3 induce ability to capture beads and proteins in human fibroblasts. (A) HDFs transduced with PIB were incubated overnight with FITC-labelled latex beads (1 μm) and analysed by fluorescent microscopy at day 7 after addition of Dox. CellVue Claret Far Red and DAPI were used to stain cellular membranes and nuclei, respectively. (B) Flow cytometry analysis of PIB-transduced HDFs after incubation with Ovalbumine-AlexaFluor647 for 20 minutes at 37° C. at day 7 after addition of Dox. Controls were kept on ice (4° C.).

FIG. 42. PIB factors induce Clec9a and MHC-II expression in lung cancer cells. Flow cytometry analysis of Clec9a and MHC-II expression in PIB-transduced 3LL cells at day 8 after addition of Dox. M2rtTA-transduced cells are included as control.

FIG. 43. PIB factors induce CLec9a and MHC-II expression in melanoma cells. Flow cytometry analysis of Clec9a and MHC-II expression in PIB-transduced B16 cells at day 8 after addition of Dox. M2rtTA-transduced cells are included as control.

FIG. 44. Delivery of PIB factors in a polycistronic vector increases reprogramming efficiency. (A) Schematic representation of polycistronic regions encoding either Pu.1, Irf8 and Batf3 (PIB) or Irf8, Pu.1 and Batf3 (IPB) separated by 2A-like sequences. (B) Flow cytometry analysis of Clec9a reporter activation in MEFs transduced with Pu.1, Irf8 and Batf3 in individual vectors (top, right panel) or polycistronic vectors (PIB and IPB) at day 7 after addition of Dox. M2rtTA-transduced cells are included as control. (C) Quantification of tdTomato+ cells after transduction with PIB factors in individual or polycistronic vectors at day 7.

DETAILED DESCRIPTION

The present disclosure relates to compositions, nucleic acid constructs, methods and kits thereof for cell induction or reprogramming cell to the dendritic cell state or antigen presenting cell state, based, in part, on the surprisingly effect described herein of novel use and combinations of transcription factors that permit induction or reprogramming of differentiated or undifferentiated cells into dendritic cells or antigen presenting cells. Such compositions, nucleic acid constructs, methods and kits can be used for inducing dendritic cells in vitro, ex vivo, or in vivo, and these induced dendritic cells or antigen presenting cells can be used for immunotherapy applications.

Natural DCs are bone marrow-derived cells that are seeded in all tissues. DCs are poised to sample the environment and to transmit the gathered information to cells of the adaptive immune system (T cells and B cells). Upon antigen engulfment, DCs initiate an immune response by presenting the processed antigen, which is in the form of peptide-major histocompatibility complex (MHC) molecule complexes, to naive (that is, antigen inexperienced) T cells in lymphoid tissues. After activation, DCs typically overexpress co-stimulatory and MHC molecules in addition to secrete various cytokines responsible for initiating and/or enhancing many T and B lymphocyte responses, i.e. type I interferon, tumor necrosis factor (TNF)-α, IFN-γ, IL-12 and IL-6. Thus, DCs are generally identified by their high expression of major histocompatibility complex class II molecules (MHC-II), co-stimulatory molecules, such as CD80/86 and CD40, and integrin CD11c, as well as their superior capacity to secrete inflammatory cytokines and to migrate from non-lymphoid to lymphoid organs and stimulate naïve T cells. In mice and humans, distinct subsets of DCs can be variably defined by phenotype, ontogeny, and function. They include the conventional DC subset 1 (cDC1, also kwon as CD8α+ DC subset) found in mouse lymphoid organs and the related CD103+ DC subset in non-lymphoid tissues. Cells bearing a similar phenotype have recently been described in humans, humanized mice, and sheep, indicating cross-species conservation of the cDC1 family. This extended family has distinct functional properties, most notably a remarkable efficiency at capturing material from dead or dying cells, as well as processing exogenous antigens for cross-presentation on MHC class I. These two features allow cDC1 DCs to cross-present cell-associated antigens and trigger CTL responses against infectious agents or tumors. In addition to priming CD8+ T cells, cDC1+ DCs have been implicated in the establishment of cross-tolerance to tissue-specific cell-associated antigens. The ability of cDC1 DCs to either cross-prime or cross-tolerize CD8+ T cells against cell-associated antigens implies that they can decode the context in which they encounter dead cells. DNGR-1, also known as CLEC9A, is a receptor for necrotic cells that favors cross-priming of CTLs to dead cell-associated antigens in mice. DNGR-1 is selectively expressed at high levels by mouse cDC1 DCs, CD103+ DCs and by their human equivalents, being responsible for recognizing an intracellular ligand exposed after cell death. Recently, expression of Clec9a was shown to allow the identification of DC precursors (CDPs) committed to the conventional DC lineage and their progeny in lymphoid tissues (10).

The successful identification of DC inducing factors capable of reprogramming differentiated cells to induced DCs, as described herein, can advance our basic understanding of DC biology in a number of ways. This work will provide thorough insight into DC minimal transcriptional networks. In addition, the identification of DC inducing factors offer unprecedented opportunities to understand how DC state is established and how key regulatory machinery is put into place.

Transcription factors play a critical role in the specification of all cell types during development. The success of direct reprogramming strategies using transcription factor-mediated reprogramming indicates that it is equally plausible to direct the differentiation of pluripotent ES/iPS cells or multipotent stem cells to specific fates using such factors. Accordingly, using the DC inducing factors identified herein, directed differentiation of ES/iPS cells to a definitive DC fate by expression of the DC-enriched transcription factors can be achieved. Additionally, using the DC inducing factors identified herein, directed differentiation of multipotent hematopoietic stem and progenitor cells to a definitive DC fate by expression of the DC-enriched transcription factors can be achieved (forcing differentiation along the hematopoietic tree depicted in FIG. 1)

Typically, nucleic acids encoding the DC inducing factors, e.g., DNA or RNA, or constructs thereof, are introduced into a cell, using viral vectors or without viral vectors, via one or repeated transfections, and the expression of the gene products and/or translation of the RNA molecules result in cells that are morphologically, biochemically, and functionally similar to DCs, as described herein. These induced DCs (iDCs) after priming with the adequate antigens have the ability to capture, process and present them to effectors cells of the immune system (macrophages, T-cells, B-cells, NK cells) eliciting antigen-specific immune responses against cancer, viral and parasitic/bacterial infections (FIG. 2).

An aspect of the present disclosure is the use of TFs or the use of a combination of TFs in cancer cells (in situ or ex vivo) to force them to present their own antigens to immune cells (FIG. 3). This method represents a feasible strategy to increase the clinical outcome of anticancer immunotherapies as it bypasses cancer evasion mechanisms and increases tumor immunogenicity.

In an embodiment, 18 candidate TFs were selected due to their specifically enriched gene expression in DCs (FIG. 4A), enriched in DCs when compared to macrophages, which are less efficient APCs (FIG. 4B, left panel) (20) and during DC ontogeny (FIG. 4B, right panel). Gene ontology (GO) enrichment analysis for the 18 TFs highlighted their fundamental role on leucocyte and DC differentiation (p=2.51E-12 and p=9.58E-12, respectively) and activation (p=1.02E-11 and p=6.31E-11, whilst mouse mutant phenotype enrichment analysis confirmed that genetic perturbations in those genes cause largely hematopoietic phenotypes, in particular abnormal adaptive immunity (p=1.19E-04) and abnormal antigen presentation (p=1.25E-03) (FIG. 4C). 18 candidate TFs were cloned individually in a reprogramming proven Doxycycline (Dox)-inducible lentiviral vector (6).

In an embodiment, for screening the effect of the new dendritic cell-inducing TFs and DC-inducing TF combinations by cellular reprogramming, it has started with Mouse Embryonic Fibroblasts (MEFs) harboring a DC-specific reporter (Clec9a-Cre X R26-stop-tdTomato) and used the activation of the reporter to shown DC-inducing TFs. In Clec9a-tomato reporter mouse, the tdTomato fluorescent protein is expressed exclusively by CDPs, pre-DCs and in cDCs (10). Macrophages, other immune lineages or monocyte-derived DCs in culture do not express Clec9a and therefore the tdTomato protein (FIG. 5A). Within the immune system Clec9a gene expression is selectively restricted to CDPs and their progeny (pre-cDCs and cDCs) (FIG. 5B). Results from gene expression analysis of cDC and precursors also highlighted that Clec9a expression is acquired after commitment to cDC lineage in CDPs and pre-DCs and not before in Monocyte DC progenitors (MDPs) (FIG. 5C) (11). Clec9a is expressed in CDPs, both pre-DCs and cDC subset, reaching high levels in the cDC1 subset (FIG. 6A) (21). Spleen cells isolated from Clec9a reporter mice were analysed, confirming that 98.8% of cDC1 cells (gated in MHC-II+CD11c+CD8a+) express the tdTomato fluorescent protein (FIG. 6B).

Double transgenic Clec9a-tdTomato reporter MEFs were isolated from E13.5 embryos and excluded from any contaminating tdTomato+ or CD45+ cell that could be already committed to the hematopoietic lineage (FIGS. 7A and 7B) by Fluorescent-Activated Cell Sorting (FACS). MEFs used for screening and in the following experiments were tdTomato− CD45− with a purity of 99.8% (FIG. 7C).

In an embodiment, PU.1, IRF8 and BATF3 are sufficient for Clec9a activation and to impose dendritic cell morphology.

In an embodiment, Clec9a reporter MEFs were transduced with combinations of candidate TFs and evaluated for tdTomato expression (FIG. 8). After transduction with the 18 candidate TFs or one of the pools of 4 TFs, we observed the emergence of tdTomato+ cells 5 days after adding Dox (FIG. 9A, FIG. 9B). The pool comprising of Pu.1, Irf4, Irf8 and Batf3 generated more tdTomato+ cells than 18 TFs (2.36% versus 0.59%, respectively) suggesting that the minimal combination of factors required to induced reporter activation is contained within this pool. TdTomato+ cells were not detected after transduction with control M2rtTA vector. We then removed each of the factors individually (FIG. 9C). Pu.1, Irf8 and Batf3 (PIB) removal reduced reporter activation while removal of Irf4 did not have an impact. These results suggest that PIB are essential for DC reprogramming.

In an embodiment when the combination of PU.1, IRF8 or BATF3 (PIB) was expressed in MEFs the Clec9-reporter is activated with an increased efficiency (approx. 3.96%, FIG. 10A). In an embodiment was then evaluated the kinetics of reporter activation (FIG. 10B). TdTomato+ cells start to be detected between day 1 and day 2 and peak between day 5 and day 7 (FIG. 10B). In an embodiment removal of PU.1, IRF8 or BATF3 completely abolished reporter activation whereas their individual expression was not sufficient to generate tdTomato+ cells (FIG. 10C). These data suggest that in this embodiment PIB constitute the minimal combination of TFs for Clec9a activation and induced Dendritic Cell (iDC) generation. Importantly, tdTomato+ cells display increased size and complexity (FIG. 10D), consistent with the observed stellate morphology and the establishment of dendrites characteristic of DCs (FIG. 10E, FIG. 10F). It has been confirmed that reporter activation occurs around 30 hours by time-lapse microscopy and observed that tdTomato+ cells exhibited morphology changes, migration capacity and dendrites gradually being established within 6 days (FIG. 11). The pan-hematopoietic marker CD45 is expressed in approximately 20% of PIB-transduced MEFs, with approximately 6.6% of tdT+ cells included in this population (FIG. 12).

In an embodiment was evaluated the impact of expressing additional factors to PIB. It was assessed the individual impact of each of the TFs from the candidate pool of 18 TFs (FIG. 13A) as well as other hematopoietic TFs (FIG. 13B). From the 31 TFs tested it was observed that STAT3, IKZF1, IRF2, NFIL3, BCL6, L-MYC, RUNX1 and KLF4 negatively impact the numbers of tdTomato+ cells generated. The addition of TCF4 and Gfi1b showed a 2.2-fold and 1.9-fold increase in reporter activation, respectively.

In an effort to optimize the culture conditions for iDC generation it was tested the addition of the cytokines Granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4 and FMS-like tyrosine kinase 3 ligand (Flt3I) during the induction (FIG. 14A) because of their important role during DC specification (12). It was also tested adding lipopolysaccharides (LPS), different media compositions (RPMI, 2-mercaptoethanol (2-ME) and 4 mM L-Glutamine (2×Glut) and co-culture with the stromal cells OP-9 and OP9-DL1 (FIG. 14B) (13). These culture modifications did not increase the number of induced tdTomato+ cells.

In an embodiment, the in vivo expression patterns of the PIB and TCF4 were analysed. PU.1, IRF8, BATF3 and TCF4 transcripts are expressed in single DC precursor cells (FIG. 15). While Pu.1 is equally expressed in MDPs, CDPs and Pre-DCs, IRF8 expression markedly increases in CDPs and is maintained in pre-DCs. BATF3 and TCF4 are only up-regulated at a later stage, in pre-DCs. Moreover, the combined expression of PU.1+IRF8+BATF3 is mostly enriched in CD8α+ DCs among 96 cells and tissues (FIG. 16A). Importantly, Pu.1 levels are higher in both pre-DC stages, while Irf8 and Batf3 are specifically enriched in pre-cDC1 and cDC1 subsets (FIG. 16B). When compared to other DC subsets and several hematopoietic cell lineages, Clec9a, Pu.1, Irf8 and Batf3 display increased expression of in CD103+ DCs belonging to cDC1 subset (FIG. 16C).

In an embodiment, it was evaluated whether the activation of the C9a-tdTomato reporter was reflected in the expression of DC markers, such as typical surface markers used to discriminate between conventional cDC and pDC subsets (FIG. 17A, FIG. 17B). Interestingly, the cDC1 marker CD103 is expressed in 25±13.65% of tdTomato+ cells in contrast to undetectable levels in tdTomato− cells suggesting the specification of a non-lymphoid migratory cDC1 program. Moreover, we could detect only residual expression of cDC2 and pDC markers (0.41±0.58% CD4+, 2.56±0.18% CD11b+, 0.77±1.09% B220+ cells, respectively).

In an embodiment was evaluated, if the activation of the Clec9a-tdTomato reporter was reflected in the expression of key components of the antigen presentation machinery at the cell surface. Remarkably it was observed that 71.4% of tdTomato+ cells at day 7 expressed MHC-II at the surface (FIG. 18A), a key molecule for the establishment of APC functionality. The expression of MHC-II is gradually acquired starting between day 1 and day 2 and peak between day 7 and day 11 (FIG. 18B). The kinetics of MHC-II activation resembles the activation of the Clec9a reporter (FIG. 10B). At day 7 the tdTomato-compartment contained a lower percentage of MHC-II+ cells (14.2%) (FIG. 18A). It was then addressed whether expression of MHC-II was controlled directly by PIB factors. By excluding each of the factors individually it was observed that PU.1 expression, but not IRF8 or BATF3 expression, is important for MHC-II activation (FIG. 18C), consistent with the described Pu.1 role in regulating Class II Transactivator (CIITA) through its promoter I (CIITApI) (14, 15). CIITA is known as the master regulator of MHC Class II genes' expression, determining cell-type-specific, cytokine-induced and developmental-derived modulation of MHC-II expression through the differential usage of CIITA promoters (16). In conventional DCs, CIITApI has been associated with regulation of MHC-II genes.

In an embodiment, due to the described involvement of IRF4 in inducing MHC-II expression through interaction with CIITA (17), it was evaluated whether IRF4 could compensate for Pu.1 in the generation of MHC-II+ cells within the tdTomato+ population. It was therefore assessed the expression of MHC-II in tdTomato+ cells generated by 4TFs (including IRF4) or their individual exclusion (FIG. 18D). Inclusion of IRF4 in the pool did not increase MHC-II expression on tdTomato+ cells and IRF4 could not substitute for the loss of Pu.1. Accordingly, IRF4 and PU.1 were found to synergistically promote MHC-II expression through CIITA promoter III in B cells but not DCs (17). During reprogramming to iDCs, no synergism with PU.1 was observed, which was strictly required for MHC-II expression in tdTomato+ cells.

In an embodiment, it was evaluated the expression of MHC class I molecules, key molecules for the establishment of APC functionality. 56.7% of tdTomato+ cells at day 7 expressed MHC-I at the surface (FIG. 19). At day 7 the tdT-compartment contained a lower percentage of MHC-I+ cells (11.2%) (FIG. 19).

In an embodiment, it was evaluated the expression the co-stimulatory molecules CD80 and CD86, required for efficient antigen presentation (FIG. 20). CD80 and CD86 are expressed in 35.2% of tdTomato+MHC-II+ cells in contrast to only 12.9% of tdTomato+ MHC-II− cells. This characterization of the expression of MHC-II, CD80 and CD86 at the cell surface of iDCs suggests that a cohort of tdTomato+ MHC-II+ cells would be competent in antigen presentation. An additional co-stimulatory molecule, CD40, is expressed in 16.1% of tdTomato+ cells, comparing to only 2.8% of tdTomato− cells (FIG. 21A). Resting cDCs, in particular cDC1 subset, have been described to respond to microbial stimulation up-regulating the expression of co-stimulatory molecules and becoming more effective APCs (25). Accordingly, tdTomato+ cells up-regulate the expression of CD40 (4-fold increase) at cell surface after toll-like receptor TLR4 stimulation (LPS) (FIG. 21B).

In an embodiment, in order to define the extent of transcriptional changes during iDC reprogramming, it was measured the full-length single-cell transcriptomes after transduction with PIB (FIG. 22A). 192 cells were initially profiled from nontransduced MEFs, sorted day 3 Clec9a-tdTomato+, day 7 Clec9a-tdTomato+, day 9 Clec9− tdTomato+MHC-II+ cells and freshly isolated CD11c+ MHC-II+ CD8α+ splenic DCs (sDCs). From these, 163 individual cells passed quality control filters and were used for analysis. After alignment of reads at individual gene loci, it has used the Census algorithm to convert relative RNAseq expression levels (transcript per million, TPM) into relative transcript counts (Census counts) without the need for experimental spike-in controls and improving the accuracy of differential expression analysis. It was first employed t-distributed stochastic linear embedding (tSNE) algorithm to perform clustering analysis of the genome-wide transcriptomes (FIG. 22B) and observed two main clusters of cells. IDCs at day 3 and the majority of day 7 cells map close to MEFs, whilst the remaining day 7 and all day 9 iDCs cluster together with sDCs. It was performed unsupervised clustering using Single-Cell Consensus Clustering (SC3), confirming the global similarity of sDCs with the group of day 9 and some of the day 7 iDCs (FIG. 22C). Moreover, the timing of global transcriptome reprogramming correlates well with the peak generation of Clec9a-tdTomato+ MHC-II+ cells between days 7 and 15 (FIG. 18B). TdTomato+ cells show time-dependent transcriptional changes starting as early as day 3; by day 9 iDCs are remarkably similar to bona fide DCs. This analysis suggests that PIB factors induce global transcription reprogramming towards the DC fate. It was extracted the 6525 most variable genes across the dataset, which after clustering could be organized in 4 groups (FIG. 22D). Cluster I comprises highly expressed genes in MEFs, which are silenced during DC reprogramming. These include typical fibroblast markers, such as Col5a2, Grem1, Lox, Acta2 and Thy1 (FIG. 22E). Cluster II includes transcripts enriched at day 3 and day 7, suggesting activation during the initial stages of reprogramming. This cluster comprises genes such as Eea1 and Aldh1a2 that are associated with intracellular trafficking and metabolism as well as type I interferon (IFN) signaling (Ifit3) (FIG. 22F). In contrast, Cluster III encompasses genes enriched at day 9 (FIG. 22D). Interestingly, MHC-II related genes (such as H2-Pb) as well as genes regulating cross-presentation in DCs (such as the cathepsin Ctsc and Cd74) are enriched in this cluster (FIG. 22F). Finally, Cluster IV includes genes enriched in sDC1s and reprogrammed iDCs (FIG. 22D), such as the pan-hematopoietic marker Cd45 and the general DC marker Cd11c (FIG. 22F). Importantly, cDC1-restricted genes were also upregulated, such as the Clec9a gene and TIr3 (22), and the key regulator of MHC class I-dependent immune responses (Nlrc5) necessary for antigen cross-presentation, a key feature of cDC1s (23). Indeed, we have detected a robust increase in cDC1-signature genes during the reprogramming process when compared to cDC2-signature genes (FIG. 26) (11). Collectively, these data suggest complete DC reprogramming favoring the cDC1 subset. Accordingly GO analysis showed that categories related to antigen processing and presentation (p-value=3.34E-08, 1.80E-06, 3.53E-06, 4.03E-06) where enriched top biological process and pathways (Table 1). Top cellular component GO categories include lysosome (p-value=1.31E-07) and lytic vacuole (p-value=1.44E-07), concordant with the described role of lysosome signaling in coordinating antigen processing and migration of DCs (24). MicroRNA (miRNA) target prediction showed highest enrichment of miR-155 (p-value=1.41E-11) and miR-124 (p-value=1.00E-10) targets (Table 2), that have been implicated in DC function and specification, respectively (25,26).

TABLE 1 Top 5 gene ontology biological process (left) and cellular component (right) enriched in Cluster I to IV. GO Biological Processes P-value GO Cellular Processes P-value Cluster I Translation 1.06E−81 Ribonucleoprotein 1.46E−96 Generation of metabolites and 1.50E−34 complex energy Ribosome 1.75E−90 Electron transport chain 2.58E−26 Mitochondrion 6.32E−72 Intracellular transport 1.52E−24 Mitochondrial part 7.38E−50 Establishment of protein 3.61E−19 Ribosomal subunit 6.27E−37 localization Cluster II Chromatin modification 2.74E−05 Membrane-enclosed 2.96E−06 Protein transport 1.07E−04 lumen Establishment of protein 1.22E−04 Intracellular organelle 5.10E−06 localization lumen Macromolecule catabolic 1.54E−04 Organelle lumen 5.53E−06 process Endoplasmatic reticulum 9.61E−06 Protein catabolic process 1.54E−04 Non-membrane bound 9.98E−05 organelle Cluster Antigen presentation of 3.34E−08 Vacuole 1.38E−08 III exogenous antigen Lysosome 1.31E−07 Antigen presentation of 1.80E−06 Lytic vacuole 1.44E−07 exogenous peptide Cytosol 3.08E−05 Actin cytoskeleton organization 2.71E−06 Endoplasmatic reticulum 4.19E−05 Antigen presentation of peptide 3.53E−06 antigen Antigen processing and 4.03E−06 presentation Cluster Chromosome organization 3.28E−09 Cytoskeleton 2.51E−11 IV Cell cycle 5.10E−09 Non-membrane bound 1.24E−09 Regulation of GTPase-mediated 9.30E−09 organelle signaling Intracellular organelle 1.24E−09 DNA metabolic process 2.64E−08 Endomembrane system 3.39E−09 Regulation Ras protein signal 1.20E−07 Microtubule cytoskeleton 2.31E−08 transduction

TABLE 2 Gene ontology mouse loss-of-function mutant phenotype (top panel), KEGG pathways (middle panel) and microRNA target interactions (bottom panel) enrichment analysis was performed on the 4 clusters of genes identified using the 6,525 most variable genes across the 5 sample groups (relative to FIG. 22). The lists show the most enriched terms and the right columns show respective p-values by fold change in relation to the top enriched term. Cluster I P-value Cluster II P-value

Mouse Abnormal cell death 2.36E−11 Abnormal response 4.25E−06 Phenotypes to infection Abnormal cell physiology 3.44E−12 Abnormal embryonic tissue 3.87E−06 Premature death 1.10E−12 Abnormal development 2.68E−06 patterning Abnormal extraembryonic 5.36E−13 Abnormal extraembryonic 1.00E−06 tissue tissue Abnormal embryonic tissue 1.86E−13 Abnormal embryo size 5.38E−07 Abnormal cell proliferation 7.69E−14 Abnormal immune system 1.38E−07 Abnormal embryo size 2.25E−14 Abnormal adaptive immunity 4.15E−08 Cellular phenotype 7.15E−19 Abnormal immune cell 3.61E−08 Mammalian phenotype 8.04E−29 Mammalian phenotype 5.99E−09 Prenatal Lethality 8.24E−50 Prenatal Lethality 5.05E−10 Pathways Citrate cycle (TCA cycle) 2.06E−05 Endocytosis 8.69E−02 Ubiquitin mediated 1.51E−05 Apoptosis 8.01E−02 proteolysis Nucleotide excision repair 8.73E−06 Vesicular transport 7.84E−02 Spliceosome 9.90E−11 Lysosome 3.79E−02 Proteasome 1.01E−14 Galactose metabolism 3.35E−02 Alzheimer’s disease 2.76E−20 RIG-I-like receptor signaling 3.35E−02 Parkinson’s disease 3.04E−31 Antigen processing/ 3.30E−02 presentation Huntington’s disease 1.33E−32 Cytosolic DNA-sensing 1.47E−02 Oxidative phosphorylation 3.72E−34 Toll-like receptor signaling 4.50E−03 Ribosome 2.32E−53 Ubiquitin mediated 3.09E−03 proteolysis MicroRNAs miR-1-3p 9.67E−17 miR-100-5p 3.85E−05 Let-7b-5p 1.91E−17 miR-7b-5p 2.78E−05 miR-320a 1.08E−18 miR-425-5p 2.63E−05 miR-484 1.72E−19 miR-19b-3p 2.07E−05 miR-92a-3p 7.25E−20 miR-98-5p 1.93E−05 miR-100-5p 1.84E−20 Let-106b-5p 4.53E−06 miR-186-5p 1.80E−20 miR-93-5p 4.39E−06 miR-30a-5p 7.02E−21 miR-215-5p 3.24E−06 miR-615-3p 1.11E−26 miR-21-5p 3.01E−06 miR-16-5p 6.33E−40 miR-192-5p 2.43E−06 P-value Mouse Phenotypes 6.36E−11 3.22E−11 2.73E−11 6.53E−12 4.54E−12 2.92E−12 5.46E−13 4.15E−13 1.40E−15 9.50E−17 Pathways 6.38E−04 6.09E−04 5.83E−04 5.06E−04 5.02E−04 4.48E−04 1.77E−04 1.33E−04 3.02E−05 2.08E−09 MicroRNAs 2.92E−07 2.76E−07 1.15E−07 5.32E−08 4.41E−08 1.54E−08 7.27E−09 4.60E−09 1.00E−10 1.41E−11 Cluster IV P-value Abnormal morphology 3.90E−15 Cellular phenotype 2.36E−15 Premature death 1.73E−16 Abnormal brain morphology 1.48E−16 Pre-natal lethality 4.28E−19 Preweaning lethality 1.80E−20 Post-natal lethality 1.64E−20 Abnormal survival 1.70E−22 Mortality/aging 7.48E−23 Mammalian phenotype 8.97E−38 Endometrial cancer 3.56E−03 Adherent junction 3.32E−03 Pancreatic cancer 1.80E−03 Pathways in cancer 1.08E−03 Long-term depression 6.48E−04 Prostate cancer 6.44E−04 Insulin signaling 1.90E−04 Inositol metabolism 1.59E−04 Acute myeloid leukemia 9.09E−05 Phosphatidylinositol signal. 1.16E−05 miR-425-5p 1.64E−06 miR-484 9.27E−07 miR-21-5p 7.15E−07 miR-223-3p 3.97E−07 miR-181a-5p 8.57E−08 miR-9-5p 1.12E−08 miR-149-5p 7.37E−10 miR-218-5p 1.85E−10 miR-340-5p 1.25E−10 miR-324-3p 1.11E−12

indicates data missing or illegible when filed

This analysis also revealed activation of DC transcriptional regulators, including Zbtb46 and Bcl11a, which were originally included in our candidate TF list (FIG. 23A). Several members of the interferon-regulatory factors IFN and signal transducer and activator of transcription (STAT) protein families were also identified. For example, Stat2 and 117, key mediators of TLR-Induced DC activation and type I IFN responses, respectively, are detected in high levels at day 3 and 7. Stat6 also display high median expression values for day 3 and day 7, whilst Stat1 expression increases at day 9 to levels 128-fold higher than splenic DCs. DC maturation has been reported to be accompanied by a change from STAT6 to STAT1 utilization, which suggests that Pu.1, Irf8 and Batf3 overexpression may also induce DC maturation. It was observed high levels of expression of Pu.1, Irf8 and Batf3 at day 3, day 7 and day 9 when compared to sDCs, consistently with the lentiviral-mediated expression of the 3 TFs (FIG. 23B, top panel). Since our lentiviral vectors encode the coding sequences without UTRs, it was quantified the expression levels of the endogenous transcripts using the reads at the 3′- and 5′-UTRs. Importantly, it was observed the expression of endogenous Pu.1, Irf8 and Batf3 starting at day 3 (FIG. 23B, bottom panel). At day 9 of reprogramming, endogenous expression levels are comparable to splenic DCs.

In order to further characterize the dynamic nature of the transcriptional reprogramming, gene set enrichment analysis (GSEA) was performed using NetPath gene sets to compare the transitions between day 0, 3, 7 and 9 (FIG. 24, top panel). It was observed that several immune-related gene sets were highly enriched in day 3 compared with MEFs. Interestingly, IL-4 used for in vitro differentiation of DCs ranked on top (NES: 1.97, FDR q-value: 0.02) (FIG. 24, bottom left panel). Some gene sets were also enriched on day 7, although with smaller NES values, suggesting that more subtle transitions might occur during this phase. In contrast, several gene sets were highly enriched in day 9 compared with day 7, including interleukin pathways and Oncostatin M, previously associated with DC maturation (FIG. 24, bottom right panel).

In an embodiment, the transcriptional networks for step-wise transitions during iDC reprogramming were evaluated (FIG. 25A). It was observed that the transition of MEFs to day 3 was associated with the expression of a dense TF network that highly connected to the PIB reprogramming factors; the transition of day 3 to day 7 was softer, characterized by a less dense TF network, which do not include the PIB factors; and the transition of day 7 to day 9, characterized by a dense TF network which can be divided in 2 clusters of TFs, one denser that includes the cDC marker Zbtb46, and one composed by fewer TFs including the PIB factors. This reinforces the idea that a subtler transition might occur between day 3 and day 7, and suggests that day 9 iDCs might have acquired a stable cell fate. Importantly, all these transcriptional regulators are enriched in mature DCs and not in DC progenitors, irrespective of the day that are activated, suggesting that the reprogramming process do not pass through an intermediate progenitor state (FIG. 25B). Moreover, the absence of intermediate progenitor cells in the iDC reprogramming process was further validated by performing hematopoietic colony formation assays with PIB-transduced MEFs at day 3, 5, 7, 10 and 25 after addition of Dox, including sorted sDC1s and unsorted splenocytes and bone marrow cells as controls (FIG. 25C). No colonies were observed in iDCs or sDC1 cultures whereas, as expected, unsorted splenocytes and bone marrow cells in culture gave origin to colonies. This supports the idea that the reprogramming process is direct and do not transit through intermediate progenitor cells.

In an embodiment, it was set out to reconstruct the DC reprogramming path by establishing a pseudo-temporal order based on the gradual transition of cell transcriptomes (FIG. 27A). Using pseudo-Time reconstruction in Single-Cell RNA-seq Analysis (TSCAN) software, it was observed that the order is consistent with the temporal reprogramming events, with MEFs being followed by day 3 iDCs and subsequently by the majority of day 7 iDCs. Interestingly, 4 individual day 7 and some day 9 iDCs are positioned in line with sDCs in the pseudotime ordering, suggesting that the transcriptome reprogramming was complete. However, the remaining day 9 iDCs seem to be further away from the initial timepoint than the splenic DCs. In order to confirm the robustness of these results, it was performed pseudo-time ordering using Monocle2, an alternative algorithm for delineating differentiation paths. This reconstruction positioned biological sample groups along 3 branches of pseudotime ordering (FIG. 27B, left panel). MEFs, d3 iDCs and 26 day 7 iDCs were placed along the first branch, considered cell state 1 (FIG. 27B, right panel), which then reaches a branching point and divides into State 2 and State 3. State 2 includes 82% of sDCs as well as 3 day 7 and 13 day 9 iDCs. However, 55% of day 9 iDCs as well as 11 sDCs are placed within State 3, which is consistent with TSCAN results. To understand the transcriptional differences between these 2 states, GO enrichment analysis was performed using the differentially expressed genes (Table 3), which showed that top biological processes and pathways in State 3 include type I and type II (IFN-γ) IFN signaling, known inflammatory mediators of DC activation and maturation (38, 39). Genetic perturbations for State 3 enriched genes highlighted corresponding immune phenotypes, such as abnormal APC and abnormal immune system. Consistently, BEAM analysis revealed 2 kinetic clusters of branch-dependent genes upregulated in State 3 (cluster 2 and 4) functionally enriched for antigen presentation and other immune-related processes (FIG. 28A and Table 4).

TABLE 3 Top 5 gene ontology biological process, mouse phenotypes and wiki pathways enrichment analysis of genes differentially expressed between State 2 and State 3. Relative to FIG. 27. State 2 P-Value State 3 P-Value GO Biological Phosphatidylinositol 5.90E−04 IFNγ signaling 2.58E−06 processes dephosphorylation Response to IFNγ 6.98E−06 Regulation of error-prone 6.50E−04 Cellular response to IFNγ 7.39E−06 translesion synthesis Neutrophil 2.21E−05 Actin filament capping 1.05E−03 degranulation Actin filament reorganization 1.19E−03 Type I interferon 2.24E−05 Peptidyl-serine 2.13E−03 signaling autophosphorylation Phenotypes Lethality at weaning 2.56E−06 Abnormal antigen 1.00E−09 Premature death 9.47E−05 presenting cell Abnormal mineral 1.25E−03 Abnormal immune 8.88E−08 homeostasis system Mammalian phenotype 9.44E−04 Abnormal blood cell 1.34E−07 Abnormal startle reflex 3.40E−03 Abnormal response to 1.40E−07 infection Abnormal bone marrow 1.79E−07 Pathways Estrogen signaling pathway 7.03E−03 IFNγ signaling 9.80E−07 Regulation of actin 9.07E−03 G13 Signaling 3.09E−06 cytoskeleton Alzheimers Disease 5.68E−06 Breast cancer 9.50E−03 Heart Hypertrophy 1.52E−05 MAPK signaling 1.06E−02 IL-3 Signaling Pathway 1.94E−05 Gastric cancer 1.06E−02

TABLE 4 Top 5 gene ontology biological process and mouse loss-of-function mutant phenotype enrichment analysis of genes in Cluster 1 to 5. Relative to FIG. 28. GO Biological processes P-value Mouse Phenotypes P-value Cluster 1 Translation 6.97E−05 Abnormal cell death 8.32E−04 DNA packaging 5.26E−03 Abnormal lacrimal gland 1.25E−03 Nucleosome assembly 1.41E−02 Abnormal sex gland 2.11E−03 Chromatin assembly 1.51E−02 Abnormal hormone levels 3.18E−03 Protein-DNA assembly 1.57E−02 Abnormal muscle 4.46E−03 contractility Cluster 2 Negative regulation signal 7.46E−03 Abnormal blood cell 2.33E−06 transduction Abnormal immune system 2.52E−05 Negative regulation cell 1.20E−02 Abnormal immune cell 7.16E−04 communication Perinatal lethality 9.04E−04 Regulation of cell killing 1.29E−02 Abnormal adaptive 6.72E−04 Regulation leukocyte 1.29E−02 immunity cytotoxicity 2.23E−02 Regulation lymphocyte immunity Cluster 3 Sensory perception chemical 9.34E−23 Mammalian phenotype 3.04E−14 stimulus Abnormal blood 9.59E−09 Neurological system process 1.04E−22 homeostasis Sensory perception 1.67E−22 Abnormal hormone levels 9.62E−09 Sensory perception smell 6.48E−22 Abnormal nervous system 7.57E−08 Cognition 2.96E−21 Abnormal neuron 6.91E−08 morphology Cluster 4 Antigen presentation of 1.94E−05 Abnormal blood cell 1.81E−09 exogenous peptide Abnormal Immune system 5.66E−08 Antigen presentation via MHC-II 1.94E−05 Abnormal antigen 1.77E−07 Polysaccharide antigen 4.03E−05 presenting cell presentation Abnormal immune cell 8.54E−07 Positive regulation leukocyte 8.20E−05 Abnormal bone marrow 1.87E−06 activation Exogenous antigen 8.91E−05 presentation Clusters 5 Protein-DNA complex assembly 4.71E−03 Mammalian phenotype 4.93E−05 Chromosome organization 7.89E−03 No abnormal phenotype 4.81E−04 Chromatin 1.01E−02 Normal phenotype 5.16E−04 Lens development in eye 1.34E−02 Metabolism phenotype 1.54E−03 Positive regulation of secretion 1.47E−02 Abnormal Social 5.30E−03 interaction

In an embodiment, GSEA also showed that 4705 vs 167 gene sets for immunological signatures were upregulated on State 3 when compared with State 2, such as Mature Stimulatory DC, IFNγ and IFNα stimulated DC gene sets (FIG. 28B). As State 3 contained the majority of day 9 iDCs, it was sought to confirm that similar maturation trait was observed when comparing sDC1s (naïve) with day 9 iDCs. GSEA showed that antigen processing and presentation and DC maturation gene sets are enriched at day 9 iDCs (FIG. 29A). Interestingly, Stat6, which is associated with immature DCs, was up regulated in sDC1s, whilst Stat1, described to increase with maturation, was up regulated in day 9 iDCs (FIG. 23A).

In an embodiment, given that it was previously observed that iDCs express high levels of MHC-II molecules, which is reported to be associated with maturation of DCs (FIG. 18), it was sought to investigate if the observed pseudotime trajectories were indicative of different maturation states. It was observed that the branch kinetic curves reflect a continuous upregulation of Ciita, the known master regulator of MHC-II genes' expression, and several MHC-II genes (H2-Aa, H2-Ab1 and H2-Eb1) towards State 3 as compared with State 2 (FIG. 30A). Consistently, expression of Ciita and genes associated with mouse (Tnfrs1a, Tapbp, Inpp5d and Traf6) and human (Acp5 and Itag4) DC maturation were enriched at day 9 iDCs (FIG. 30B). These data suggest that iDCs are intrinsically more mature than sDCs and may be less dependent on exogenous activation stimuli for antigen presentation.

In an embodiment, the induced dendritic cells in some aspects of all the embodiments of disclosure, while similar in functional characteristics, differ in their gene expression from the naturally occurring endogenous dendritic cells (Table 5).

TABLE 5 Top 500 differentially expressed genes between day 9 iDCs and sDC1 cells ordered by fold change. Day 9 up Day9 Down (vs sDC1) Fold change (vs sDC1) Fold Change Cd74 8.378400519 AY036118 3.630967107 Ucp2 7.999114666 Sfi1 3.546674749 Grn 7.650480054 Gprc5c 3.440084658 Cdkn1a 7.079169574 Olfr648 3.00881167 S100a11 7.048233116 Rsph9 2.959047864 Gapdh 6.979316778 Tmsb4x 2.890522248 Cct8 6.923788452 Mtmr1 2.654214297 Ly6e 6.336631107 Il15 2.574244187 Ubb 6.19368097 Fanci 2.24583185 B2m 6.151779369 Ptprk 2.113646962 Mir6240 6.053040315 Cnot6l 2.002604494 Irf8 5.725731065 Bdkrb1 1.987001179 Mir6236 5.674808976 Pdgfb 1.930540437 Spi1 5.640038385 Letm1 1.915941208 Cd81 5.563653083 Abca3 1.880254741 Ctsa 5.501860486 Plpp5 1.867877278 Rnf13 5.457727173 1700095J03Rik 1.847936727 Ifitm3 5.400810698 Dcun1d4 1.788829125 Samhd1 5.263523757 Lrif1 1.769132531 Pgam1-ps2 5.203763065 Fus 1.758030571 Prkar1a 5.192511183 Ltbp1 1.719160475 Gns 5.151976827 Prr15l 1.702342332 Gdi2 5.020765205 Rps20 1.686771039 mt-Nd5 4.991318969 Fam92a 1.682146576 Usp14 4.895884787 Rpl32 1.661123211 Eif2ak3 4.883252352 Clstn3 1.658201639 mt-Tm 4.874702287 Usp10 1.651957116 Med21 4.82652063 2900009J06Rik 1.642513164 Shisa5 4.762756365 4930553l04Rik 1.639560609 Stat1 4.717469627 Nceh1 1.633238968 Scpep1 4.654917481 Kdelr1 1.623624988 Tmem59 4.630781554 Amdhd2 1.620291498 Drg1 4.622644784 Snhg14 1.60518724 Pttg1ip 4.613419142 Ppcdc 1.593210807 Batf3 4.567419599 Cit 1.571609014 Grina 4.563608382 Lef1 1.571116453 Ctsc 4.562081251 Cinp 1.555760027 Calm2 4.5319627 Cep290 1.542877805 Ifi44 4.526513658 Eya4 1.542329932 Arhgdib 4.448903084 Ssbp2 1.541337751 Ifitm2 4.43505433 Stard3nl 1.53918705 Itm2b 4.41388282 Ppp2r5a 1.529811517 Sbds 4.328188964 Rps27 1.514118829 Bst1 4.322914109 Rpain 1.512378029 Nnat 4.253027937 Rpsl5a 1.503115843 Sulf2 4.233589183 Ushbp1 1.489434725 Lgals3bp 4.212602771 Caprin2 1.48766573 Dazap2 4.138256756 Glis1 1.468306086 Slc30a9 4.045135818 Rpl24 1.455561714 Rbms1 4.026259385 Rgs1 1.452134922 Ftl1 4.022138158 Lsp1 1.445858249 Eif3a 4.002306157 Malat1 1.441188932 Axl 3.997197448 Kctd19 1.439866506 Cited2 3.993185485 Sfxn5 1.438464054 Lars2 3.932022979 Brca2 1.429734511 Cfl1 3.902855666 Fgd4 1.429251043 Pfn1 3.862256545 Mir762 1.42527755 Ate1 3.846471162 Rabgap1 1.418468643 Myadm 3.828349737 Notch1 1.417303263 Bgn 3.827554891 Anapc5 1.415567561 Ywhab 3.794470188 Slc6a17 1.412700849 mt-Rnr1 3.783136169 Ncapd2 1.407349715 Tnfrsf1a 3.708506639 Ccdc40 1.382403998 Tmbim6 3.677054824 4930519L02Rik 1.382401843 Ppp3r1 3.659734805 Aacs 1.378156309 Cap1 3.650896891 Arhgef40 1.377963701 Sparc 3.634708576 Olfr986 1.376630166 Tgtp2 3.628901251 Dnm1 1.369144578 Chordc1 3.627931997 Adgrv1 1.36678555 Mir8114 3.606630393 Reg2 1.36266606 Tma7 3.600667604 Kif24 1.360757058 Slc25a3 3.59535779 Khk 1.354650399 Unc93b1 3.541471457 Camk2d 1.353347692 Mapk1 3.539792266 Disp1 1.337935937 Spp1 3.53813155 Msh3 1.330172804 Trim25 3.537049716 Pmf1 1.328658766 Ywhaz 3.533742941 Mrpl48 1.320851411 Pla2g7 3.518104367 Fry 1.318521603 Cyfip1 3.485665307 Adgra3 1.315125368 Ncoa4 3.473391046 Tssc1 1.313253438 Tgtp1 3.454518062 Fbf1 1.312311242 Pros1 3.417372505 Hsd3b2 1.311309901 Dda1 3.40525625 Snord57 1.309355259 Cmtr1 3.387409585 Adsl 1.308071098 Stt3a 3.385900731 Banp 1.307751088 mt-Cytb 3.381159175 Diexf 1.303869281 Edem1 3.367122678 Ctage5 1.296409102 Lgmn 3.355164538 Olfr1089 1.295835162 Serinc3 3.35283067 Arhgef19 1.293919043 Plbd2 3.337813415 Trpm4 1.291888876 Slfn5 3.330993072 Olfr980 1.290974033 Rab1b 3.324732402 Ap3b2 1.290262191 Ap3d1 3.315238282 Ndufs1 1.282010137 Icaml 3.293730801 Prob1 1.279252518 Mdh1 3.270873537 Tox 1.277128111 Hsp90aa1 3.26235373 Tnk2 1.275196071 Zmpste24 3.256672868 Pcdh15 1.274945531 mt-Rnr2 3.227837673 Use1 1.273360442 Sp100 3.20878081 Znrf1 1.273285759 Surf4 3.208326066 Il16 1.265024831 Pkm 3.190910497 Gsn 1.264820606 Ciita 3.181654915 Tyrobp 1.254979854 Glul 3.165849559 Zfp57 1.25469807 Cmc4 3.145763373 Dnm3 1.249092237 Ifit2 3.140223266 Btbd19 1.246939299 AA474408 3.12954966 Tmeff2 1.244721598 Pigt 3.127377903 Pde1c 1.241625438 Serinc1 3.116484011 Slc16a14 1.241236737 Lyn 3.111346193 Herc4 1.240940968 Tnfaip1 3.108541206 Pdzd2 1.240932703 Rnf145 3.108426516 Cenpu 1.239620462 Ubl5 3.09022469 Ccdc9 1.237773411 Map2k4 3.089586057 Cd63 1.235857882 Hmga1 3.069250123 Scrib 1.229623359 Cox6a1 3.069034679 Lats2 1.222817428 Laptm4a 3.060750646 Plbd1 1.220326423 Psmc5 3.047865774 1700030K09Rik 1.212538771 Plekho2 3.039215546 Gpsm3 1.212322462 Lgals9 3.035253178 Rasa4 1.212195177 Mlxip 3.031480133 Jade1 1.211805352 Fos 2.995472377 Astn1 1.206888241 Fkbp10 2.995150234 Abca2 1.205733273 Gps2 2.993152938 Ptpn5 1.2051801 Tecr 2.987574318 Tpk1 1.201688574 Mbnl1 2.96248689 Kantr 1.201432145 Sez6l 2.951436702 Slc15a2 1.199012945 Sh3bp2 2.949222732 Fgf1 1.196064338 Mov10 2.935927814 4930511M06Rik 1.193287597 Fam167a 2.91423349 A430010J10Rik 1.193177327 Asns 2.907465523 Pde4c 1.186996538 C530025M09Rik 2.889428766 2610203C20Rik 1.183842191 Zdhhc5 2.875076576 Cep85 1.182775307 Rpl38 2.871058843 Auts2 1.181857217 Tmod3 2.869947135 Pld1 1.179419755 Tspan9 2.868408159 Lsamp 1.179262613 Arhgap1 2.861787688 Ercc5 1.174619478 Rn7s1 2.857560775 B230216N24Rik 1.172531942 Rn7s2 2.857560775 Cdkal1 1.172233483 St3gal5 2.850664779 Pib1 1.170618218 Lamp2 2.838622934 Ttc3 1.169286246 Zfp36l1 2.828619356 Smarcad1 1.1661693 Qsox1 2.816348613 B130055M24Rik 1.164407791 Nubp2 2.801535041 Glrb 1.162769632 Dap 2.798447222 Fam71e1 1.161503582 H2-Eb1 2.790275212 Actr1b 1.160650079 Trpc4ap 2.790223774 Fam120c 1.159839707 Ptk2 2.774986716 Lamb3 1.1544153 Litaf 2.76910688 Rpl18 1.147822978 Samd12 2.756415888 B430219N15Rik 1.147593372 Hspa8 2.738492847 Pnpla7 1.146410398 Psap 2.725482169 Myo1b 1.145796458 Rap1b 2.708283782 Tmcc1 1.145088425 Rab11b 2.707195739 Mxd1 1.142557058 Kdm5c 2.659888994 Gtpbp4 1.140348186 Atp6vOc 2.652904455 Mbtd1 1.139896426 Pnkp 2.627212937 Mir101c 1.139676828 Atp1b3 2.57433971 Srcin1 1.138957445 Plin2 2.569589842 C130026l21Rik 1.13879651 Dusp1 2.564304737 Qrich1 1.135006953 Dennd6a 2.55766128 Snora7a 1.129825352 Syt9 2.54591498 P2ry2 1.12826657 Park7 2.538892352 Tle6 1.127997617 Thbs1 2.526658864 Gstt2 1.127976061 Ndel1 2.520436416 Rnf214 1.125647634 Eif2s2 2.517791626 Mier2 1.125490293 Degs1 2.516228756 Rad52 1.123514249 Pcsk6 2.508442547 Tsfm 1.121124616 Washc4 2.501207547 Rp134 1.118504821 H2-Pb 2.494182352 Ampd2 1.118010861 Gins4 2.480528831 Col6a6 1.11565181 Ap2m1 2.479969069 6820408C15Rik 1.11415417 Stam 2.460346325 Plekha7 1.11413288 Calm1 2.459547652 Kifc1 1.113349023 Cd47 2.452322667 Entpd8 1.11138608 Arhgap5 2.436336219 Trmu 1.111070484 Msn 2.427359 Dhx30 1.110688069 Arhgef2 2.424471636 Becn1 1.108510602 Rnps1 2.421957675 Gtf2h1 1.107621797 Agpat3 2.420651749 Dennd2d 1.107444842 Hexb 2.406950442 St14 1.10685259 Jmjd1c 2.403960577 Sema6c 1.105449952 Uba7 2.393024725 Olfr1321 1.105249776 Stat3 2.392854797 Nin 1.104494546 Aqr 2.390209543 2900076A07Rik 1.103729098 Rasgrp3 2.389626162 Alg13 1.102785341 Ifi207 2.378045514 Rapgef3 1.101622786 Tcn2 2.366636661 Trps1 1.101516996 Jkamp 2.362648719 Morc2a 1.100797248 Xpnpep2 2.36068485 Myo15b 1.097398552 Pld4 2.345478622 Dlg1 1.096480818 Csnk1a1 2.333344842 Pus7 1.092393911 Cmklr1 2.331529539 H2afz 1.091796066 mt-Co1 2.330383946 Cacna1f 1.091419504 Commd7 2.308633577 Rps7 1.091078271 Gabarap 2.303502443 Kctd15 1.087832781 Aes 2.298975567 Slc22a15 1.083405985 Nfe2l1 2.296002657 Nbr1 1.082686573 Sgpl1 2.2917436 Cd27 1.081589891 Gbf1 2.287585646 Itga2b 1.080996825 Gstm1 2.280980951 Eci2 1.080130803 Mtcl1 2.275467469 Cd6 1.080089799 Vcl 2.249503311 Mical1 1.080032007 Slc25a5 2.249170226 Serpina6 1.079365549 2610507B11Rik 2.249033444 Cadm2 1.079157707 Tmem248 2.243289507 Kmt5b 1.075999202 Chd9 2.24028321 Scn8a 1.075722849 B4galt5 2.23488477 Zfp239 1.075383073 Rictor 2.234163721 Ap1s1 1.075103048 Srrm2 2.233556776 Erdr1 1.071138918 Sh3bgrl 2.232075277 Cacna1a 1.069720493 Cdc42se1 2.209710506 Ivns1abp 1.067892703 Lrp1 2.208956871 Dhodh 1.067584269 Ipo9 2.195716865 Ttn 1.067147365 Tcp1 2.194202192 Ddx19a 1.066321446 Ppp1cb 2.188862064 Stx4a 1.064577603 Pgam1 2.187328026 Safb2 1.063265347 Atp6v0e 2.185473454 Aloxe3 1.063249994 Pik3r1 2.177807689 Stx18 1.062644106 mt-Nd4l 2.170033442 Notch4 1.062408256 Ddx5 2.1526024 Vars2 1.06236208 n-R5-8s1 2.137222743 Ces5a 1.06224942 Ddost 2.137080797 Fxyd2 1.061290421 Btf3 2.136870648 Olfr539 1.061124921 Pitpna 2.130897293 Ubl4a 1.06102377 Zfp451 2.121640696 Plxnc1 1.059510561 Msrb3 2.108023782 Tars2 1.059081003 Tram1 2.103942132 A430073D23Rik 1.058179534 Gpx1 2.092125019 Brpf1 1.058170111 Rab3il1 2.091780481 E2f6 1.05716641 Anxa3 2.082860042 Gle1 1.055917907 Prkaa1 2.07920508 Clca3a2 1.053662602 Rab8b 2.076422781 Mir99ahg 1.052669091 Srpr 2.060721132 Grk2 1.052345211 Ncstn 2.048033241 Firre 1.052005979 mt-Nd2 2.046373118 Cp 1.049988888 Sf3b1 2.04486528 Cacna2d4 1.04830389 H2-Ab1 2.037298163 Isy1 1.046348457 Plp2 2.021912335 4930402H24Rik 1.046335717 Dnm1l 2.011874745 D430042O09Rik 1.045493524 Nptx1 2.007694218 Pax6 1.043231341 Clec16a 1.998844774 Rcbtb2 1.042445583 Uggt1 1.98850241 Wapl 1.042368448 Pxk 1.982701164 Rab14 1.040736807 Tiparp 1.982534756 Magi3 1.038947888 Impad1 1.982393464 Ctbp2 1.037976024 Gpcpd1 1.979316308 Prpf4b 1.037971623 Tmem214 1.973030883 Csmd2 1.037185914 Coro1b 1.970528885 Btbd11 1.036685875 Naaa 1.968987323 Vwf 1.036683252 Snx12 1.960818344 Cdh4 1.036640143 Anpep 1.959292302 Apbb1 1.036638533 Ptk2b 1.94897573 Ccdc162 1.0359892 Gusb 1.944307923 Sipa1l3 1.035923762 Ccnd3 1.940195368 Slc35a3 1.035192913 Syf2 1.939540412 Ahrr 1.034311688 Tubb5 1.927300046 Opcml 1.034020412 Ap2b1 1.927092864 Sirt5 1.032682041 Col4a1 1.92298865 Nox4 1.032270813 Myl12b 1.918466916 Spint2 1.031953157 Ccnd1 1.912610788 Aebp2 1.031681705 Sfxn3 1.906741793 Dtnbp1 1.030548993 Timp2 1.906668629 Iqca 1.030543359 B230219D22Rik 1.902284133 Lbp 1.029703558 Rhog 1.887582293 Kcnj16 1.029523684 Scap 1.88592784 Gtf2ird1 1.028843965 Qk 1.88561085 Aldoa 1.026887502 Bfar 1.882090302 Pfdn5 1.026732581 Slfn5os 1.880082558 Mtx3 1.026090598 Lipa 1.879981666 Zfp950 1.025199024 Plekha1 1.876160326 Rasgrp4 1.024647963 Errfi1 1.86913871 Lmf1 1.024604555 Ccnd2 1.866791989 Smc3 1.024558747 Snhg4 1.85926484 Fam118b 1.024505164 Zmat3 1.858777231 Kif15 1.023953631 Ptpn9 1.856452888 Cpeb3 1.023923019 Egr1 1.853897687 Adgra1 1.023026376 Dnajc10 1.852044296 Safb 1.022908183 A630033H20Rik 1.851287962 Psmb3 1.022863235 Ctsb 1.832770858 Dhtkd1 1.022813101 Sgsh 1.831682811 Bmpr1b 1.02276876 Ctnna1 1.825509641 Cdk14 1.022075321 Gng12 1.823669353 Abcd3 1.020692022 Tmem176b 1.82285949 A530040E14Rik 1.019535471 Atp6v0a2 1.820322631 Phf24 1.018368684 Dmd 1.814015876 Frmd5 1.017848005 Ssbp4 1.808520665 Cldn34c1 1.017608358 Dck 1.807120995 Mfap4 1.017464779 Tmed10 1.804630829 Lgi1 1.016790218 Plekha2 1.788241201 Fgfr2 1.016403211 Ywhae 1.787342486 Espn 1.015617966 Prdx6 1.782181966 Olfr90 1.015595827 Cpne8 1.780683612 Ahcyl2 1.015502202 Pan3 1.76632335 Zbtb46 1.015224258 Tsn 1.765727062 Ghrhr 1.013691524 Postn 1.760918581 Slu7 1.013465906 5031439G07Rik 1.754798139 Rgs6 1.011093407 Tcf25 1.751773197 Hacl1 1.010778823 Capza2 1.748806651 Myo1g 1.01023387 Ssr3 1.745487096 Tsen54 1.00985654 Pafah1b1 1.741737246 Tdo2 1.006294884 Sbf2 1.740917993 Mrgprc2-ps 1.006158745 Ubc 1.738762972 Sez6 1.005579581 Rnpep 1.733612821 Fmr1 1.004972266 Tnpo1 1.73276259 Olfr295 1.004922065 1110037F02Rik 1.731726851 Stard10 1.004561273 Ogt 1.722820519 Ikzf3 1.003969279 Nras 1.722695811 Mad1l1 1.003331901 Ddx39b 1.722321035 Sun2 1.002634026 Elovl5 1.716649083 Zfp532 1.001725437 Inpp5d 1.708655113 C2cd3 1.001540813 Stx7 1.705989329 Gpr89 1.001332809 Klf3 1.704318719 C920009B18Rik 1.001138066 Sdc3 1.691630397 Itsn1 1.001091655 PItp 1.689657257 BC034090 1.000446333 Gnai2 1.686128128 Gripap1 0.999489556 Nfib 1.677548572 Lmo7 0.998665132 Eef1a1 1.666817084 Cep250 0.99700681 Sval2 1.654461632 Mkln1 0.996736898 Cxcl16 1.653930195 9030624J02Rik 0.996673208 Gpr108 1.649897733 C2cd5 0.994435489 Atp5h 1.648363833 Racgap1 0.994262554 Ppp1ca 1.648036693 Epb41 0.993574572 Amfr 1.646430431 Rgs3 0.9935682 2310014F06Rik 1.642609288 Map2k2 0.991526748 mt-Tl1 1.638589779 Zfp369 0.990602507 Twsg1 1.636545598 Zcchc4 0.990232518 Magt1 1.631891466 Celf3 0.989871179 Gria3 1.614777482 Nfrkb 0.988761687 Gna12 1.611782252 1500012K07Rik 0.987475187 Ppp4r1l-ps 1.611024994 Csnk1g1 0.987302343 Mfge8 1.606938172 Tbk1 0.987242185 Lasp1 1.606641944 Ube2e2 0.986976054 Gstp1 1.60150536 C2cd2l 0.986252848 Sh3pxd2b 1.598640187 Nlgn1 0.985395138 Coq10b 1.597748785 Atad3aos 0.98523759 Cdk1 1.590738694 Lair1 0.98518503 Wnk1 1.589077278 Lamtor3 0.983751037 Calm3 1.575849963 Man2c1 0.98191137 Rad23b 1.575037653 Phc2 0.981299938 Naa20 1.570197421 Rnf123 0.980776141 Nkx2-2 1.566605188 Rgs11 0.980226854 Nfix 1.555019024 Fbxo18 0.980008734 Nans 1.547167513 Plxna3 0.979857188 Sidt2 1.545962364 Adam23 0.979186928 Oasl2 1.532383232 Thsd7a 0.979160545 Cyb561a3 1.53078711 Pde4d 0.979053978 Rasal2 1.530399343 Smim1 0.977702838 Flt4 1.527570552 Pum2 0.977669452 2810474O19Rik 1.526141876 Dlec1 0.977609037 3222401L13Rik 1.520827618 Arhgef4 0.977585828 Fyttd1 1.520231944 Zbtb49 0.977502295 ligp1 1.515968737 Senp3 0.977451995 Atp6vla 1.514613918 Trpm2 0.976880999 Lrrc42 1.513457922 1810032O08Rik 0.976754675 Trim16 1.513078049 Gramd1c 0.976420001 Tmub2 1.511718079 Zfp13 0.975991964 Slc25a12 1.511137106 Ppp4r1 0.975773873 Oasl1 1.509642511 Proser2 0.975505959 Rpph1 1.507834495 Nek10 0.975435395 Crtc3 1.506271977 Mcf21 0.974595726 Rnf44 1.502558854 Cald1 0.974287067 Rab43 1.4997319 Homez 0.973060396 Lrch4 1.494344133 Plcg1 0.972958606 Trim35 1.493644106 Pkp4 0.972945011 Slit2 1.489830377 Hnrnpk 0.972631359 Cyp2f2 1.488373517 Ppp2r1a 0.97246017 Snx3 1.481595088 Trmt1 0.972303044 Etv5 1.479921021 Rab3gap1 0.972187837 Oas2 1.473261403 4930431F12Rik 0.972082764 Psme1 1.470561263 Tcte2 0.97206615 Lsm12 1.468223249 Aoah 0.9714557 Impact 1.466855064 1700110l01Rik 0.970367518 Dcakd 1.465419814 4933427J07Rik 0.970106045 Tbp 1.463219259 Polrmt 0.969835731 Alg8 1.463180722 Plekhg3 0.969499396 Csrp1 1.460388067 Chrna9 0.969212179 Znfx1 1.459793233 Fgfr1op2 0.969013346 Ctps 1.457382832 Olfr889 0.968607339 Zc3h14 1.456860742 Gnas 0.968283741 Nisch 1.454173681 Egflam 0.967634053 Polr2a 1.45089857 Clk4 0.966015603 Hectd1 1.443602365 Metap1d 0.965868178 Mir195b 1.442373355 Rap1gap 0.965285567 Rnf139 1.439425895 Inpp5f 0.965246376 Hist1h4m 1.436213948 Olfr509 0.964350171 Yap1 1.436000261 Trpml 0.964078371 Cse1l 1.432919664 Palm 0.963865503 Hist1h4n 1.420524579 Capn10 0.96369017 Lhx9 1.412383525 Acad10 0.962450168 Plekhnl 1.410987625 Xndcl 0.962213076 Arpc4 1.404917958 Tesk2 0.961846014 Vamp3 1.404006857 Acox2 0.961694735 Phkb 1.402306049 Ptpn3 0.960945148 Atp1a1 1.398979263 Slf1 0.960893662 Scamp2 1.393517046 Rpl23 0.95970086 Rnf213 1.392440751 Hdac7 0.959267451 Grb10 1.389174949 Prkcb 0.958836304 Znrf2 1.388534731 Bcas3 0.958813276 Hspa5 1.385032431 Rpl19-ps10 0.958779312 Dnase2a 1.37456459 Efcab7 0.958287456 Cyr61 1.371301936 Pabpc1 0.95824206 Cystm1 1.370152464 Rassf8 0.95818526 Hnrnpl 1.35916454 Lrmp 0.957724619 Ppia 1.357022789 1700034P13Rik 0.957400444 Pptc7 1.353998906 Rspry1 0.95567678 Fxr1 1.347297931 Sorbs2 0.955664718 Kif1c 1.345550329 Rtel1 0.955325539 Ctsd 1.339661798 Snph 0.955256444 Tgoln1 1.338617781 Clk1 0.952095381 Fam65a 1.336440467 Tdh 0.951361116 Synpo 1.335052229 4930571N24Rik 0.95129822 Fbrs 1.332797189 Frmd4b 0.951044409 Abcc1 1.330281279 Txnrd2 0.950976492 Ranbp2 1.330010856 D10Wsu102e 0.950757437 Ubr4 1.327808199 Stxbp2 0.950655783 Sel1l 1.325945409 Mum1 0.950334319 Tsg101 1.322854408 Adam12 0.949845685 Bag1 1.32115605 Gramd1b 0.949409727 Cmtm3 1.319131098 Duxbl1 0.94925517 Rsu1 1.318682847 Pmaip1 0.949000209 Il6st 1.318295734 Fance 0.948894855 Gng2 1.315481592 Prose 0.948788392 Tmem184b 1.31525017 Lima1 0.948520832 Gatm 1.314698511 Aen 0.94849098 Mir1193 1.31441673 Prdm16 0.948418105 Pias1 1.314346226 Pcca 0.948384277 Elk3 1.309805838 4933411E06Rik 0.948034366 Rnf130 1.301482051 Slc26a4 0.947701486 Rpl13 1.300399066 Dgkd 0.946864762 Lpp 1.296602519 Csnk1e 0.945641621 Mrpl45 1.296477005 Katnal2 0.945160424 Cyb5r3 1.296253036 Vcam1 0.944460807 Shprh 1.289669952 Tmem200a 0.944191277 Cpt1c 1.289477514 Chek2 0.943761214 Ptpn1 1.282425471 Sgk2 0.943639303 Fam160a2 1.279579655 Nsun5 0.943217275 Cfh 1.26988982 Tcf7l1 0.941235147 Hnrnpul1 1.266397252 Uckl1 0.941137918 Txndc12 1.266168771 Rasgrp2 0.941009198 Eri3 1.264671312 Smarcd2 0.940938344 Gsk3b 1.256436011 Epha7 0.939472345 Rnh1 1.255232223 Armc6 0.938877158 Man1b1 1.250578892 Ptpn22 0.938849214 Fkbp1a 1.245105748 Fev 0.93824194 Mia3 1.244304037 Serpinb6a 0.938234426 Ruvbl2 1.239887848 Mier1 0.938199749 Adam10 1.233094367 4930567H12Rik 0.938007376 Mfap5 1.232219563 Sgsm1 0.936969063 Trim56 1.226423401 Csn1s1 0.936473012 Aaed1 1.225534965 Herpud1 0.936370431 Mapre1 1.223804151 Braf 0.936045549 Laptm5 1.223212463 Npsr1 0.935924917 Glipr2 1.21815041 Cox6b1 0.935454199 Dock10 1.213163554 Dpm1 0.935059488 Tmx2 1.211625711 Rhoa 0.935000601 Tor1aip2 1.210578968 Chrnb3 0.934196093 Etv6 1.208482154 Cobl 0.934107222 Vmn1r70 1.208124474 Al838599 0.933705762 Anxa5 1.205547419 Vars 0.932030709 Cuta 1.203534407 Clspn 0.931082728 Larp1 1.202436659 Dvl2 0.930782278 Tapbp 1.19794257 Dync1h1 0.930369494 Ddx6 1.193876061 Luc7l2 0.930153333 Mbnl2 1.186460159 Rell2 0.929377802 Ncoa3 1.179909627 Zfp260 0.929183972 Tpm3 1.179006377 Dock3 0.929117216 Dok1 1.178638573 Bace1 0.928792603 Per1 1.177725503 Sh3gl2 0.927925558 Prrc2b 1.177576351 Pde1a 0.927007191 Memo1 1.173534294 Zfyve1 0.925770343 Pcbp1 1.173138043 Tacc2 0.925726245 Ccs 1.172429114 Col16a1 0.925278858 F11r 1.168823701 Urod 0.924496139 Mmp23 1.167621462 Kntc1 0.924441575 Ssr2 1.167292126 Tprn 0.92401978 Pmpcb 1.16244984 Ipmk 0.923602568 Mtmr2 1.161744403 Tns4 0.923451301 Atxn10 1.15851223 Zfp512b 0.923242144 Glg1 1.156216287 Rnf10 0.922947177 Fndc3a 1.156036991 Pus10 0.922597079 Zdhhc13 1.153699404 Slc39a9 0.922555226 Mef2c 1.150436919 Arhgap22 0.921849391 Mir8116 1.144949195 Mknk1 0.921395425 Slc6a6 1.143929269 1810059H22Rik 0.921384279 Dmpk 1.143512387 Ttc29 0.921368016 Prr32 1.137396445 Grip1 0.921036891 Zcchc6 1.136983879 Nudt16 0.920946667 Pfkp 1.136528919 Sf3b5 0.920695962 1600014C10Rik 1.134651281 Sbno1 0.920236532 Pdhb 1.13293917 4933407L21Rik 0.92002246 Elk4 1.130558075 Bend5 0.919256559 Casp3 1.129817521 Pard3 0.918710037 Gskip 1.129732393 Fam81a 0.918653108 Dnase1l3 1.127323549 Abcg2 0.918618185 Pde4b 1.124393895 Flt3 0.917955107 B4galt1 1.124344501 Nebl 0.917936388 Ube2v1 1.120565671 Ddc 0.917229801 Ifi213 1.120530139 Lrrc8d 0.916139547 Cops8 1.119841447 Focad 0.915711203 Sf3a2 1.118870719 Tlk2 0.915663628

In an embodiment, in addition to the membrane associated co-stimulatory molecules, mature DCs express cytokines with a pro-inflammatory function that are important for the development of T-cell responses. These responses can be initiated by the triggering of at least 11 different Toll-like receptors (TLRs), allowing the specific recognition of distinct conserved microbial or viral structures. It was asked whether iDCs secrete cytokines to the media when challenged with TLR3 (using Polyinosinic-polycytidylic acid (poly-I:C)) or TLR4 (Lipopolysaccharides (LPS)) stimulation (FIG. 31). Upon LPS or polyI:C challenge of iDCs it was observed an increase in the secretion of IL-6 (14- or 10-fold, respectively). An increase in the secretion of tumor necrosis factor TNF (7-fold) and interferon IFN-γ (2-fold) was also observed after LPS stimulation or polyI:C, respectively. Cells transduced with PIB plus TCF4 (PIBT) respond equivalently to stimulation displaying increased secretion of IL-6, TNF and IFN-γ. Importantly, upon stimulation of iDCs it was not observed increase in the secretion of the anti-inflammatory cytokine IL-10. These results suggest that iDCs underwent maturation towards a proinflammatory profile.

In an embodiment, it was evaluated the capacity of iDCs to mount an antigen-specific immune response. First it was evaluated whether iDCs would be able to engulf particles by incubation with 1 μm FITC-labeled latex beads. After incubation tdTomato+ cells contained numerous fluorescent beads in the cytoplasm (FIG. 32), suggesting that iDCs have established the competence for phagocytosis.

Then, we evaluated the ability of iDCs to capture soluble proteins. Remarkably, 13.8% of tdTomato+ cells were able to actively uptake soluble protein after incubation at 37° C. for 20 minutes in contrast to only 5.6% when incubated at 4° C. (FIG. 33A), further suggesting that iDCs have established the competence for phagocytosis/endocytosis. In contrast, only 4.6% of tdTomato− cells showed similar ability. Next, we evaluated whether iDCs were able to internalize dead cell material in vitro (FIG. 33B-D). This unique ability has been shown to be associated with cDC1 DC subtypes to cross-present cell-associated antigens on MHC-I (27). After overnight incubation with labeled dead cells, 65.7% of purified tdTomato+ cells have incorporated dead cell material in contrast to only 10.5% of tdTomato-(FIG. 33B). Uptake of dead cells was further analysed by live imaging and it was observed that tdTomato+ cells avidly accumulated dead cell material in the cytoplasm (FIG. 33C). TdTomato+ cells move actively and, upon encountering a dead cell, projected cellular protrusions to incorporate and engulf it (FIG. 33D).

Moreover, we have confirmed that iDCs express genes encoding TLR (TIr3 and TIr4) and other mediators of TLR signaling, including MyD88-dependent (TRAM (encoded by Ticam2), and Traf6) and independent (MICE) pathways (FIG. 34A). Also, we have confirmed that iDCs express key mediators of receptor-mediated endocytosis (Fcgr2b, Tfr2 and Mrc1) and macropinocytosis of dead cells (Axl, Lrp1 and Scarf1), further suggesting that iDCs have acquired the ability for sense and incorporate antigens (FIG. 34B).

In an embodiment, it was evaluated the functional capacity of iDCs to promote antigen-specific proliferation of CD4 T-cells (FIG. 35A). For this it was employed MHC class II-restricted ovalbumin-specific T cells (OT-11 cells) isolated from lymph nodes and spleen of OT-II Rag2 KO mice (18). In this model T cells respond to the processed antigenic peptide (OVA 323-339) when shown in the context of MHC-II of DCs. Therefore OT-II CD4 T-cells were co-cultured with iDCs when given the Ovalbumin protein (OVA) or pre-processed antigenic peptide (OVA 323-339). Functional DCs are able to capture the protein, process and present the processed antigenic peptide in the context of MHC-II. Induced CD4+ T cell proliferation was measured by CFSE dilution and the activation of the T-cell activation marker CD44 after 7 days of co-culture. Remarkably, 56% of CD4+ T cells diluted CFSE when co-cultured with PIB-generated iDCs in the presence of OVA protein (FIG. 35B). When co-cultured with MEFs transduced with PIB+TCF4, 38.2% of CD4+ T cells diluted CFSE content, which suggests that inclusion of TCF4 in the reprogramming pool does not increase the stimulatory ability of iDCs. Splenic MHC-II+CD11c+ DCs were used as controls and generated 24.1% of proliferative T-cells. Importantly, addition of LPS stimuli, which is commonly employed to induce DC “maturation”, increased the antigen-specific stimulatory ability of MEFs transduced with PIB (1.5-fold) or PIB+TCF4 (2-fold) and also splenic DCs (3-fold) (FIG. 35B and FIG. 36). As expected, T-cells that were not co-cultured did not proliferate with or without LPS stimuli. To further assess the stimulatory ability of iDCs, it was evaluated if they were able to induce expression of T-cell activation markers, such as CD44. When given the pre-processed antigen, OT-II CD4 T cells diluted CFSE and upregulated the expression of CD44 when co-cultured with both PIB-generated iDCs and splenic MHC-II+CD11c+ DCs (FIG. 35C). Importantly, when given OVA protein, iDCs display comparable ability to induce CD44 expression in OT-II T cells when compared with splenic DCs (52.2% versus 63.8%). This data supports iDCs' functional ability to incorporate and process OVA protein followed by presentation of processed Ovalbumin peptides in MHC-II complexes at cell surface. Collectively, these results highlight the functional capacity of iDCs and support the feasibility of using directly reprogrammed fibroblast to present antigens and inducing antigen-specific adaptive immune responses.

In an embodiment, it was evaluated if iDCs acquire ability to export antigens to cytosol and express key genes essential for cross-presentation ability. Cross-presentation via the cytosolic pathway involves antigen export from endocytic compartments to the cytosol. Thus, the ability of iDCs to perform antigen export was evaluated using a cytofluorimetry-based assay (FIG. 37A). Remarkably, after 90-minute incubation with b-lactamase, approximately 80% of CCF4-loaded iDCs expressed cleaved CCF4. Thus, iDCs were able to uptake b-lactamase and efficiently export it into the cytoplasm, leading to the generation of cleaved CCF4. It was also confirmed that iDCs express genes involved in cross-presentation pathway, such as Cybb, Atg7, Tap1 and Tap2 (FIG. 37B).

In an embodiment, it was evaluated if iDCs were able to cross-present antigens to CD8+ T-cells. For this, cross-presentation of OVA at MHC-I molecules was evaluated by co-culturing iDCs with B3Z T-cell hybridoma cells that express β-galactosidase under the control of IL-2 promoter (FIG. 38A, left panel). It was observed that iDCs were able to induce antigen-specific T-cell activation in a concentration-dependent manner. Moreover, it was observed an increase of activation of B3Z T-cells after TLR3 stimulation with polyI:C and not with LPS (FIG. 38A, right panel). Accordingly, it has been described that maturation of cDC1s with polyI:C enhances the MHC-I cross-presentation process (28). Moreover, cross-presentation was also evaluated employing MHC class I-restricted ovalbumin-specific T cells (OT-1 cells) isolated from lymph nodes and spleen of OT-1 Rag2 KO mice (19). In this model T-cells respond to the processed antigenic peptide (OVA 257-264) when shown in the context of MHC-1 of DCs. Therefore OT-I CD8 T-cells were co-cultured with iDCs in the presence of the Ovalbumin protein (OVA) and polyI:C stimulation (FIG. 38B). Functional DCs are able to capture the exogenous protein, process and perform cross-presentation of the processed antigenic peptide in the context of MHC-1, inducing activation of CD8+ T cells. Induced CD8+ T cell proliferation was measured by CFSE dilution and the activation of the T-cell activation marker CD44 after 4 days of co-culture. Remarkably, in the presence of OVA protein, 11.3±1.13% of CD8+ T cells diluted CFSE and up-regulated CD44 expression when co-cultured with iDCs, respectively (FIG. 38B). Splenic MHC-II+CD11c+ DCs were used as controls and generated 18.05±0.78% of proliferative and CD44+ T-cells.

In an embodiment, Human Dermal Fibroblasts (HDFs) were transduced with PIB (FIG. 39A). Importantly, it was observed morphologic alterations when PIB TFs were introduced in HDFs. Three days after transduction it was observed that HDFs lost the characteristic bipolar and elongated shapes and acquired a stellate DC-like morphology (FIGS. 39B and 39C). Moreover, it was evaluated the expression of typical DC surface markers. Human foreskin fibroblasts Bis transduced with PIB express HLA-DR molecules and CLEC9A, known specific human DC markers (FIG. 40), suggesting that PIB combination of DC-inducing factors is conserved between the mouse and human and is sufficient to generate human iDCs.

In an embodiment, it was evaluated whether human iDCs would be able to engulf particles by incubation with 1 μm FITC-labeled latex beads. After incubation PIB-transduced HDFs contained numerous fluorescent beads in the cytoplasm (FIG. 41A), suggesting that iDCs have established the competence for phagocytosis. Additionally, the ability to incorporate proteins was evaluated by incubating iDCs with AlexaFluor647-labelled ovalbumine (FIG. 41B). After incubation at 37° C., 1.2% of PIB-transduced HDFs contained labelled protein, suggesting that iDCs are able to actively engulf proteins.

In an embodiment, it was evaluated whether cancer cells would acquire DC phenotypic traits after transduction with PU.1, IRF8 and BATF3. Remarkably, 2.13% of PIB-transduced lung cancer cells (3LL cell line) expressed MHC-I molecules and 2.33% co-expressed MHC-II and CLEC9A at cell surface 8 days after addition of Dox (FIG. 42). Similarly, 26% of PIB-transduced B16 melanoma cancer cells expressed CLEC9A, whilst 1.04% of cells co-expressed it with MHC-II molecules (FIG. 43). These results suggest that it is possible to induce DC phenotype in cancer cells using PIB factors.

In an embodiment, coding regions of PU.1, IRF8 and BATF3 were cloned into polycistronic inducible lentiviral vectors that express the three nucleic acid sequences, each of them separated by 2A peptide sequences (FIG. 44A). The 3 TFs were included in different orders, PU.1, IRF8 and BATF3 (PIB) or IRF8, PU.1 and BATF3 (IPB). Impressively, Clec9a reporter activation was observed in 10.8% and 6.6% of MEFs transduced with the polycistronic vectors (PIB and IPB, respectively). In contrast, only 1.9% of tdTomato+ cells were observed in MEFs transduced with lentiviral vectors encoding individual factors (FIGS. 44B and 44C). As expected, delivery of PIB factors in polycistronic vectors increased reprogramming efficiency up to 6-fold.

In an embodiment, coding regions of each candidate TF were individually cloned into an inducible lentiviral pFUW-TetO vector (6) in which the expression of the TFs is under the control of the tetracycline operator and a minimal CMV promoter. A previously described lentiviral vector containing the reverse tetracycline transactivator M2rtTA under the control of a constitutively active human ubiquitin C promoter (FUW-M2rtTA) was used in combination. Human Embryonic Kidney (HEK) 293T cells were transfected with a mixture of TF-encoding plasmids, packaging constructs and the VSV-G envelope protein. Viral supernatants were harvested after 36, 48 and 60 hours, filtered (0.45 μm, Corning) and used fresh or concentrated 40-fold with Amicon ultra centrifugal filters (Millipore).

In some embodiments, polypeptide variants or family members having the same or a similar activity as the reference polypeptide encoded by the sequences provided in the sequence list can be used in the compositions, methods, and kits described herein. Generally, variants of a particular polypeptide encoding a DC inducing factor for use in the compositions, methods, and kits described herein will have at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.

Homo sapiens Basic Leucine Zipper ATF-Like Transcription Factor (BATF3), mRNA (SEQ. ID. 1) and a codon optimized, or different codons encoding the same amino acids, are naturally also contemplated to be covered by the reference to the nucleic acid as set forth herein.

Homo sapiens Spi-1 proto-oncogene (PU.1), mRNA (SEQ. ID. 7) and a codon optimized, or different codons encoding the same amino acids, are naturally also contemplated to be covered by the reference to the nucleic acid as set forth herein.

Homo sapiens Interferon Regulatory Factor 8 (IRF8), mRNA (SEQ. ID. 5) and a codon optimized, or different codons encoding the same amino acids, are naturally also contemplated to be covered by the reference to the nucleic acid as set forth herein.

Homo sapiens Transcription factor 4 (TCF4), mRNA (SEQ. ID. 13) and a codon optimized, or different codons encoding the same amino acids, are naturally also contemplated to be covered by the reference to the nucleic acid as set forth herein.

In some embodiments of the compositions, methods, and kids provided herein, the number of DC inducing factors used or selected to generate iDCs from a starting somatic cell, such as a fibroblast cell or hematopoietic lineage cell, a multipotent stem cell, an induced pluripotent stem cell, a cancer or tumor cell is at least three. In some embodiments, the number of DC inducing factors used or selected is at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least thirty, at least thirty three, at least thirty five, at least forty, or more.

Also provided herein, in various aspects of the compositions, methods, and kits, are isolated amino acid sequences, and isolated DNA or RNA nucleic acid sequences encoding one or more DC inducing factors for use in making iDCs.

In some embodiments of the compositions, methods, and kits described herein, the nucleic acid sequence or construct encoding the DC inducing factor(s), such as PU.1, IRF8, BATF3 and TCF4, is inserted or operably linked into a suitable expression vector for transfection of cells using standard molecular biology techniques. As used herein, a “vector” refers to a nucleic acid molecule, such as a dsDNA molecule that provides a useful biological or biochemical property to an inserted nucleotide sequence, such as the nucleic acid constructs or replacement cassettes described herein. Examples include plasmids, phages, autonomously replicating sequences (ARS), centromeres, and other sequences that are able to replicate or be replicated in vitro or in a host cell, or to convey a desired nucleic acid segment to a desired location within a host cell. A vector can have one or more restriction endonuclease recognition sites (whether type I, II or IIs) at which the sequences can be cut in a determinable fashion without loss of an essential biological function of the vector, and into which a nucleic acid fragment can be spliced or inserted in order to bring about its replication and cloning. Vectors can also comprise one or more recombination sites that permit exchange of nucleic acid sequences between two nucleic acid molecules. Vectors can further provide primer sites, e.g., for PCR, transcriptional and/or translational initiation and/or regulation sites, recombination signals, replicons, additional selectable markers, etc. A vector can further comprise one or more selectable markers suitable for use in the identification of cells transformed with the vector.

In some embodiments of the compositions, methods, and kits described herein, the expression vector is a viral vector. Some viral-mediated expression methods employ retrovirus, adenovirus, lentivirus, herpes virus, pox virus, and adeno-associated virus (AAV) vectors, and such expression methods have been used in gene delivery and are well known in the art.

In some embodiments of the compositions, methods, and kits described herein, the viral vector is a retrovirus. Retroviruses provide a convenient platform for gene delivery. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells of the subject either in vivo or ex vivo. A number of retroviral systems have been described. See, e.g., U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-90; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-52; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-37; Boris-Lawrie and Temin (1993) Curr. Opin. Genet. Develop. 3:102-09. In some embodiments of the compositions, methods, and kits described herein, the retrovirus is replication deficient. Retroviral vector systems exploit the fact that a minimal vector containing the 5′ and 3′ LTRs and the packaging signal are sufficient to allow vector packaging, infection and integration into target cells, provided that the viral structural proteins are supplied in trans in the packaging cell line. Fundamental advantages of retroviral vectors for gene transfer include efficient infection and gene expression in most cell types, precise single copy vector integration into target cell chromosomal DNA and ease of manipulation of the retroviral genome.

In some embodiments of the compositions, methods, and kits described herein, the viral vector is an adenovirus-based expression vector. Unlike retroviruses, which integrate into the host genome, adenoviruses persist extrachromosomally, thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57:267-74; Bett et al. (1993) J. Virol. 67:5911-21; Mittereder et al. (1994) Human Gene Therapy 5:717-29; Seth et al. (1994) J. Virol. 68:933-40; Barr et al. (1994) Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-29; and Rich et al. (1993) Human Gene Therapy 4:461-76). Adenoviral vectors infect a wide variety of cells, have a broad host-range, exhibit high efficiencies of infectivity, direct expression of heterologous genes at high levels, and achieve long-term expression of those genes in vivo. The virus is fully infective as a cell-free virion so injection of producer cell lines is not necessary. With regard to safety, adenovirus is not associated with severe human pathology, and the recombinant vectors derived from the virus can be rendered replication defective by deletions in the early-region 1 (“E1”) of the viral genome. Adenovirus can also be produced in large quantities with relative ease. Adenoviral vectors for use in the compositions, methods, and kits described herein can be derived from any of the various adenoviral serotypes, including, without limitation, any of the over 40 serotype strains of adenovirus, such as serotypes 2, 5, 12, 40, and 41. The adenoviral vectors used herein are preferably replication-deficient and contain the DC inducing factor of interest operably linked to a suitable promoter.

In some embodiments of the compositions, methods, and kits described herein, the nucleic acid sequences encoding the DC inducing factor(s), such as, PU.1, IRF8, BATF3 and TCF4 are introduced or delivered using one or more inducible lentiviral vectors. Control of expression of DC inducing factors delivered using one or more inducible lentiviral vectors can be achieved, in some embodiments, by contacting a cell having at least one DC inducing factor in an expression vector under the control of or operably linked to an inducible promoter, with a regulatory agent (e.g., doxycycline) or other inducing agent. When using some types of inducible lentiviral vectors, contacting such a cell with an inducing agent induces expression of the DC inducing factors, while withdrawal of the regulatory agent inhibits expression. When using other types of inducible lentiviral vectors, the presence of the regulatory agent inhibits expression, while removal of the regulatory agent permits expression. As used herein, the term “induction of expression” refers to the expression of a gene, such as a DC inducing factor encoded by an inducible viral vector, in the presence of an inducing agent, for example, or in the presence of one or more agents or factors that cause endogenous expression of the gene in a cell.

In some embodiments of the aspects described herein, a doxycycline (Dox) inducible lentiviral system is used. Unlike retroviruses, lentiviruses are able to transduce quiescent cells making them amenable for transducing a wider variety of hematopoietic cell types. For example, the pFUW-tetO lentivirus system has been shown to transduce primary hematopoietic progenitor cells with high efficiency.

In some embodiments of the methods described herein, the nucleic acid sequences encoding the DC inducing factor(s), such as PU.1 (SEQ. ID. 7, SEQ. ID. 8), IRF8 (SEQ. ID. 5, SEQ. ID. 6), BATF3 (SEQ. ID. 1, SEQ. ID. 2) and/or TCF4 (SEQ. ID. 13, SEQ. ID. 14), are introduced or delivered using a non-integrating vector (e.g., adenovirus). While integrating vectors, such as retroviral vectors, incorporate into the host cell genome and can potentially disrupt normal gene function, non-integrating vectors control expression of a gene product by extra-chromosomal transcription. Since non-integrating vectors do not become part of the host genome, non-integrating vectors tend to express a nucleic acid transiently in a cell population. This is due in part to the fact that the non-integrating vectors are often rendered replication deficient. Thus, non-integrating vectors have several advantages over retroviral vectors including, but not limited to: (1) no disruption of the host genome, and (2) transient expression, and (3) no remaining viral integration products. Some non-limiting examples of non-integrating vectors for use with the methods described herein include adenovirus, baculovirus, alphavirus, picornavirus, and vaccinia virus. In some embodiments of the methods described herein, the non-integrating viral vector is an adenovirus. Other advantages of non-integrating viral vectors include the ability to produce them in high titers, their stability in vivo, and their efficient infection of host cells.

Nucleic acid constructs and vectors for use in generating iDCs in the compositions, methods, and kits described herein can further comprise, in some embodiments, one or more sequences encoding selection markers for positive and negative selection of cells. Such selection marker sequences can typically provide properties of resistance or sensitivity to antibiotics that are not normally found in the cells in the absence of introduction of the nucleic acid construct. A selectable marker can be used in conjunction with a selection agent, such as an antibiotic, to select in culture for cells expressing the inserted nucleic acid construct. Sequences encoding positive selection markers typically provide antibiotic resistance, i.e., when the positive selection marker sequence is present in the genome of a cell, the cell is sensitive to the antibiotic or agent. Sequences encoding negative selection markers typically provide sensitivity to an antibiotic or agent, i.e., when the negative selection marker is present in the genome of a cell, the cell is sensitive to the antibiotic or agent.

Nucleic acid constructs and vectors for use in making iDCs in the compositions, methods, and kits thereof described herein can further comprise, in some embodiments, other nucleic acid elements for the regulation, expression, stabilization of the construct or of other vector genetic elements, for example, promoters, enhancers, TATA-box, ribosome binding sites, IRES, as known to one of ordinary skill in the art.

In some embodiments of the compositions, methods, and kits described herein, the DC inducing factor(s), such as PU.1 (SEQ. ID. 7, SEQ. ID. 8), IRF8 (SEQ. ID. 5, SEQ. ID. 6), BATF3 (SEQ. ID. 1, SEQ. ID. 2) and/or TCF4 (SEQ. ID. 13, SEQ. ID. 14), are provided as synthetic, modified RNAs, or introduced or delivered into a cell as a synthetic, modified RNA, as described in US Patent Publication 2012-0046346-A1, the contents of which are herein incorporated by reference in their entireties. In those embodiments where synthetic, modified RNAs are used to reprogram cells to iDCs according to the methods described herein, the methods can involve repeated contacting of the cells or involve repeated transfections of the synthetic, modified RNAs encoding DC inducing factors, such as for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, or more transfections.

In addition to one or more modified nucleosides, the modified mRNAs for use in the compositions, methods, and kits described herein can comprise any additional modifications known to one of skill in the art and as described in US Patent Publications 2012-0046346-A1 and 20120251618A1, and PCT Publication WO 2012/019168. Such other components include, for example, a 5′ cap (e.g., the Anti-Reverse Cap Analog (ARCA) cap, which contains a 5′-5′-triphosphate guanine-guanine linkage where one guanine contains an N7 methyl group as well as a 3′-O-methyl group; caps created using recombinant Vaccinia Virus Capping Enzyme and recombinant 2′-O-methyltransferase enzyme, which can create a canonical 5′-5′-triphosphate linkage between the 5′-most nucleotide of an mRNA and a guanine nucleotide where the guanine contains an N7 methylation and the ultimate 5′-nucleotide contains a 2′-O-methyl generating the Cap1 structure); a poly(A) tail (e.g., a poly-A tail greater than 30 nucleotides in length, greater than 35 nucleotides in length, at least 40 nucleotides, at least 45 nucleotides, at least 55 nucleotides, at least 60 nucleotide, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides, at least 1000 nucleotides, or more); a Kozak sequence; a 3′ untranslated region (3′ UTR); a 5′ untranslated region (5′ UTR); one or more intronic nucleotide sequences capable of being excised from the nucleic acid, or any combination thereof.

The modified mRNAs for use in the compositions, methods, and kits described herein can further comprise an internal ribosome entry site (IRES). An IRES can act as the sole ribosome binding site, or can serve as one of multiple ribosome binding sites of an mRNA. An mRNA containing more than one functional ribosome binding site can encode several peptides or polypeptides, such as the DC inducing factors described herein, that are translated independently by the ribosomes (“multicistronic mRNA”). When nucleic acids are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the invention include without limitation, those from picornaviruses (e.g. FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SW) or cricket paralysis viruses (CrPV).

In some embodiments of the compositions, methods, and kits described herein, the synthetic, modified RNA molecule comprises at least one modified nucleoside. In some embodiments of the compositions, methods, and kits described herein, the synthetic, modified RNA molecule comprises at least two modified nucleosides.

In some embodiments of the compositions, methods, and kits described herein, the modified nucleosides are selected from the group consisting of 5-methylcytosine (5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxy uridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2,N2,7-trimethylguanosine (m2,2,7G), and inosine (I). In some embodiments, the modified nucleosides are 5-methylcytosine (5mC), pseudouracil, or a combination thereof.

Modified mRNAs need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structures can exist at various positions in the nucleic acid. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) can be located at any position(s) of a nucleic acid such that the function of the nucleic acid is not substantially decreased. A modification can also be a 5′ or 3′ terminal modification. The nucleic acids can contain at a minimum one and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides.

In some embodiments, it is preferred, but not absolutely necessary, that each occurrence of a given nucleoside in a molecule is modified (e.g., each cytosine is a modified cytosine e.g., 5-methylcytosine, each uracil is a modified uracil, e.g., pseudouracil, etc.). For example, the modified mRNAs can comprise a modified pyrimidine such as uracil or cytosine. In some embodiments, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the nucleic acid are replaced with a modified uracil. It is also contemplated that different occurrences of the same nucleoside can be modified in a different way in a given synthetic, modified RNA molecule. The modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the nucleic acid may be replaced with a modified cytosine. The modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures) (e.g., some cytosines modified as 5mC, others modified as 2′-O-methylcytosine or other cytosine analog). Such multi-modified synthetic RNA molecules can be produced by using a ribonucleoside blend or mixture comprising all the desired modified nucleosides, such that when the RNA molecules are being synthesized, only the desired modified nucleosides are incorporated into the resulting RNA molecule encoding the DC inducing factor.

In certain embodiments it is desirable to intracellularly degrade a modified nucleic acid introduced into the cell, for example if precise timing of protein production is desired. Thus, in some embodiments of the compositions, methods, and kits described herein, provided herein are modified nucleic acids comprising a degradation domain, which is capable of being acted on in a directed manner within a cell.

While it is understood that iDCs can be generated by delivery of DC inducing factors in the form of nucleic acid (DNA or RNA) or amino acid sequences, in some embodiments of the compositions, methods, and kits described herein, iDC induction can be induced using other methods, such as, for example, by treatment of cells with an agent, such as a small molecule or cocktail of small molecules, that induce expression one or more of the DC inducing factors.

Detection of expression of DC inducing factors introduced into cells or induced in a cell population using the compositions, methods, and kits described herein, can be achieved by any of several techniques known to those of skill in the art including, for example, Western blot analysis, immunocytochemistry, and fluorescence-mediated detection.

In order to distinguish whether a given combination of DC inducing factors has generated iDCs, one or more DC activities or parameters can be measured, such as, in some embodiments, differential expression of surface antigens. The generation of induced DCs using the compositions, methods, and kits described herein preferably causes the appearance of the cell surface phenotype characteristic of endogenous DCs, such as CLEC9A, MHC-I, MHC-II, CD40, CD80, CD86, CD103, for example.

DCs are most reliably distinguished from other immune cells by their functional behavior. Functional aspects of DC phenotypes, or dendritic cell activities, such as the ability of a dendritic cell to induce antigen specific T cell responses, can be easily determined by one of skill in the art using routine methods known in the art, and as described herein, for example, in the Drawings, i.e., FIGS. 1-44. In some embodiments of the aspects described herein, functional assays to identify reprogramming factors can be used. For example, in some embodiments, antigen presentation and antigen cross-presentation assays can be used to confirm antigen-specific induction of T cell responses (antigen presentation potential) of iDCs generated using the compositions, methods, and kits thereof. Cytokine secretion can be used to confirm immune-modulatory properties of iDCs generated using the compositions, methods, and kits described herein. Ability to engulf particles, proteins and dead cells of iDCs generated using the compositions, methods, and kits described herein can be evaluated by culturing transduced cells in the presence of labelled beads, ovalbumine or dead cells, followed by flow cytometry analysis, respectively.

As used herein, “cellular parameter,” “DC parameter,” or “antigen presentation activity” refer to measureable components or qualities of endogenous or natural DCs, particularly components that can be accurately measured. A cellular parameter can be any measurable parameter related to a phenotype, function, or behavior of a cell. Such cellular parameters include, changes in characteristics and markers of a DC or DC population, including but not limited to changes in viability, cell growth, expression of one or more or a combination of markers, such as cell surface determinants, such as receptors, proteins, including conformational or posttranslational modification thereof, lipids, carbohydrates, organic or inorganic molecules, nucleic acids, e.g. mRNA, DNA, global gene expression patterns, etc. Such cellular parameters can be measured using any of a variety of assays known to one of skill in the art. For example, viability and cell growth can be measured by assays such as Trypan blue exclusion, CFSE dilution, and 3H-thymidine incorporation. Expression of protein or polypeptide markers can be measured, for example, using flow cytometric assays, Western blot techniques, or microscopy methods. Gene expression profiles can be assayed, for example, using RNA-sequencing methodologies and quantitative or semi-quantitative real-time PCR assays. A cellular parameter can also refer to a functional parameter or functional activity. While most cellular parameters will provide a quantitative readout, in some instances a semi-quantitative or qualitative result can be acceptable. Readouts can include a single determined value, or can include mean, median value or the variance, etc. Characteristically a range of parameter readout values can be obtained for each parameter from a multiplicity of the same assays. Variability is expected and a range of values for each of the set of test parameters will be obtained using standard statistical methods with a common statistical method used to provide single values.

In some embodiments of the compositions, methods, and kits described herein, additional factors and agents can be used to enhance iDC reprogramming. For example, factors and agents that modify epigenetic pathways can be used to facilitate reprogramming into iDCs.

Essentially any primary somatic cell type can be used for producing iDCs or reprogramming somatic cells to iDCs according to the presently described compositions, methods, and kits. Such primary somatic cell types also include other stem cell types, including pluripotent stem cells, such as induced pluripotent stem cells (iPS cells); other multipotent stem cells; oligopotent stem cells; and (5) unipotent stem cells. Some non-limiting examples of primary somatic cells useful in the various aspects and embodiments of the methods described herein include, but are not limited to, fibroblast, epithelial, endothelial, neuronal, adipose, cardiac, skeletal muscle, hematopoietic or immune cells, hepatic, splenic, lung, circulating blood cells, gastrointestinal, renal, bone marrow, and pancreatic cells, as well as stem cells from which those cells are derived. The cell can be a primary cell isolated from any somatic tissue including, but not limited to, spleen, bone marrow, blood, brain, liver, lung, gut, stomach, intestine, fat, muscle, uterus, skin, spleen, endocrine organ, bone, etc. The term “somatic cell” further encompasses, in some embodiments, primary cells grown in culture, provided that the somatic cells are not immortalized. Where the cell is maintained under in vitro conditions, conventional tissue culture conditions and methods can be used, and are known to those of skill in the art. Isolation and culture methods for various primary somatic cells are well within the abilities of one skilled in the art.

In some embodiments of these aspects and all such aspects described herein, the somatic cell is a fibroblast cell.

In some embodiments of these aspects and all such aspects described herein, the somatic cell can be a hematopoietic lineage cell.

In some embodiments of these aspects and all such aspects described herein, the somatic cell can be a cancer cell or a tumor cell.

In some embodiments of the compositions, methods, and kits described herein, a somatic cell to be reprogrammed or made into an iDC cell is a cell of hematopoietic origin. As used herein, the terms “hematopoietic-derived cell,” “hematopoietic-derived differentiated cell,” “hematopoietic lineage cell,” and “cell of hematopoietic origin” refer to cells derived or differentiated from a multipotent hematopoietic stem cell (HSC). Accordingly, hematopoietic lineage cells for use with the compositions, methods, and kits described herein include multipotent, oligopotent, and lineage-restricted hematopoietic progenitor cells, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, and lymphocytes (e.g., T-lymphocytes, which carry T-cell receptors (TCRs), B-lymphocytes or B cells, which express immunoglobulin and produce antibodies, NK cells, NKT cells, and innate lymphocytes). As used herein, the term “hematopoietic progenitor cells” refer to multipotent, oligopotent, and lineage-restricted hematopoietic cells capable of differentiating into two or more cell types of the hematopoietic system, including, but not limited to, granulocytes, monocytes, erythrocytes, megakaryocytes, and lymphocytes B-cells and T-cells. Hematopoietic progenitor cells encompass multi-potent progenitor cells (MPPs), common myeloid progenitor cells (CMPs), common lymphoid progenitor cells (CLPs), granulocyte-monocyte progenitor cells (GMPs), and pre-megakaryocyte-erythrocyte progenitor cell. Lineage-restricted hematopoietic progenitor cells include megakaryocyte-erythrocyte progenitor cells (MEP), ProB cells, PreB cells, PreProB cells, ProT cells, double-negative T cells, pro-NK cells, pre-granulocyte/macrophage cells, granulocyte/macrophage progenitor (GMP) cells, and pro-mast cells (ProMCs). A differentiation chart of the hematopoietic lineage is provided at FIG. 1.

Cells of hematopoietic origin for use in the compositions, methods, and kits described herein can be obtained from any source known to comprise these cells, such as fetal tissues, umbilical cord blood, bone marrow, peripheral blood, mobilized peripheral blood, spleen, liver, thymus, lymph, etc. Cells obtained from these sources can be expanded ex vivo using any method acceptable to those skilled in the art prior to use in with the compositions, methods, and kits for making iDCs described herein. For example, cells can be sorted, fractionated, treated to remove specific cell types, or otherwise manipulated to obtain a population of cells for use in the methods described herein using any procedure acceptable to those skilled in the art. Mononuclear lymphocytes may be collected, for example, by repeated lymphocytophereses using a continuous flow cell separator as described in U.S. Pat. No. 4,690,915, or isolated using an affinity purification step of CLP method, such as flow-cytometry using a cytometer, magnetic separation, using antibody or protein coated beads, affinity chromatography, or solid-support affinity separation where cells are retained on a substrate according to their expression or lack of expression of a specific protein or type of protein, or batch purification using one or more antibodies against one or more surface antigens specifically expressed by the cell type of interest. Cells of hematopoietic origin can also be obtained from peripheral blood. Prior to harvest of the cells from peripheral blood, the subject can be treated with a cytokine, such as e.g., granulocyte-colony stimulating factor, to promote cell migration from the bone marrow to the blood compartment and/or promote activation and/or proliferation of the population of interest. Any method suitable for identifying surface proteins, for example, can be employed to isolate cells of hematopoietic origin from a heterogeneous population. In some embodiments, a clonal population of cells of hematopoietic origin, such as lymphocytes, is obtained. In some embodiments, the cells of hematopoietic origin are not a clonal population.

Further, in regard to the various aspects and embodiments of the compositions, methods, and kits described herein, a somatic cell can be obtained from any mammalian species, with non-limiting examples including a murine, bovine, simian, porcine, equine, ovine, or human cell. In some embodiments, the somatic cell is a human cell. In some embodiments, the cell is from a non-human organism, such as a non-human mammal.

In general, the methods for making iDCs described herein involve culturing or expanding somatic cells, such as cells of hematopoietic origin, in any culture medium that is available and well-known to one of ordinary skill in the art. Such media include, but are not limited to, Dulbecco's Modified Eagle's Medium® (DMEM), DMEM F12 Medium®, Eagle's Minimum Essential Medium®, F-12K Medium®, Iscove's Modified Dulbecco's Medium®, RPMI-1640 Medium®, and serum-free medium for culture and expansion of DCs. Many media are also available as low-glucose formulations, with or without sodium. The medium used with the methods described herein can, in some embodiments, be supplemented with one or more immunostimulatory cytokine. Commonly used growth factors include, but are not limited to, G-CSF, GM-CSF, TNF-α, IL-4, the Flt-3 ligand and the kit ligand. In addition, in preferred embodiments, the immunostimulatory cytokine is selected from the group consisting of the interleukins (e.g., IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, IL-9, IL-10, IL-12, IL-18, IL-19, IL-20), the interferons (e.g., IFN-α, IFN-β, IFN-γ), tumor necrosis factor (TNF), transforming growth factor-β (TGF-β), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), the Flt-3 ligand and the kit ligand.

Cells in culture can be maintained either in suspension or attached to a solid support, such as extracellular matrix components or plating on feeder cells, for example. Cells being used in the methods described herein can require additional factors that encourage their attachment to a solid support, in some embodiments, such as type I and type II collagen, chondroitin sulfate, fibronectin, “superfibronectin” and fibronectin-like polymers, gelatin, poly-D and poly-L-lysine, thrombospondin and vitronectin. In some embodiments, the cells are suitable for growth in suspension cultures. Suspension-competent host cells are generally monodisperse or grow in loose aggregates without substantial aggregation. Suspension-competent host cells include cells that are suitable for suspension culture without adaptation or manipulation (e.g., cells of hematopoietic origin, such as lymphoid cells) and cells that have been made suspension-competent by modification or adaptation of attachment-dependent cells (e.g., epithelial cells, fibroblasts).

In some embodiments of these aspects and all such aspects described herein, the isolated induced dendritic cells (iDCs) further comprise a pharmaceutically acceptable carrier for administration to a subject in need.

Also provided herein, in some aspects, are methods of treating a subject in need of treatment to induce antigen-specific immune responses to eliminate cancer cells or infectious agents using the DC inducing compositions and methods of preparing iDCs described herein, or using the isolated induced dendritic cells (iDCs) and cell clones thereof produced using any of the combinations of DC inducing factors, DC inducing compositions, or methods of preparing iDCs described herein. In such methods of treatment, somatic cells, such as fibroblast cells or hematopoietic lineage cells, can first be isolated from the subject, and the isolated cells transduced or transfected, as described herein with a DC inducing composition comprising expression vectors or synthetic mRNAs, respectively. The isolated induced dendritic cells (iDCs) produced using any of the combinations of DC inducing factors, DC inducing compositions, or methods of preparing iDCs described herein, can then be administered to the subject, such as via systemic injection of the iDCs to the subject.

Also provided herein, in some aspects, are methods of treating a subject in need of treatment to induce antigen-specific immune responses to eliminate cancer cells or infectious agents using the DC inducing compositions and any of the combinations of DC inducing factors described herein. In such methods of treatment, cancer cells are transduced, as described herein with a DC inducing composition comprising expression vectors. Cancer cells can be first isolated from the subject, transduced with a DC inducing composition comprising expression vectors and then administered to the subject, such as via systemic injection. Alternatively, cancers cells can be transduced in situ or in vivo with DC inducing composition comprising viral expression vectors. The modified cancer cell acquires antigen presentation ability, presenting their tumor antigens to T cells and eliciting cytotoxic responses against themselves.

The reprogrammed iDCs generated using the compositions, methods, and kits described herein can, in some embodiments of the methods of treatment described herein, be used directly or administered to subjects in need of immunotherapies. Accordingly, various embodiments of the methods described herein involve administration of an effective amount of a iDC or a population of iDCs, generated using any of the compositions, methods, and kits described herein, to an individual or subject in need of a cellular therapy. The cell or population of cells being administered can be an autologous population, or be derived from one or more heterologous sources. Further, such iDCs can be administered in a manner that permits them to migrate to lymph node and activate effector T cells.

A variety of means for administering cells to subjects are known to those of skill in the art. Such methods can include systemic injection, for example, i.v. injection, or implantation of cells into a target site in a subject. Cells may be inserted into a delivery device which facilitates introduction by injection or implantation into the subject. Such delivery devices can include tubes, e.g., catheters, for injecting cells and fluids into the body of a recipient subject. In one preferred embodiment, the tubes additionally have a needle, e.g., through which the cells can be introduced into the subject at a desired location. The cells can be prepared for delivery in a variety of different forms. For example, the cells can be suspended in a solution or gel or embedded in a support matrix when contained in such a delivery device. Cells can be mixed with a pharmaceutically acceptable carrier or diluent in which the cells remain viable.

Accordingly, the cells produced by the methods described herein can be used to prepare cells to treat or alleviate several cancers and tumors including, but not limited to, breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma, and the like.

In addition to the above, the methods of the invention can be used to prevent or eliminate infection by pathogens known to predispose to certain cancers. Pathogens of particular interest for use in the cancer vaccines provided herein include the hepatitis B virus (hepatocellular carcinoma), hepatitis C virus (heptomas), Epstein Barr virus (EBV) (Burkitt lymphoma, nasopharynx cancer, PTLD in immunosuppressed individuals), HTLVL (adult T cell leukemia), oncogenic human papilloma viruses types 16, 18, 33, 45 (adult cervical cancer), and the bacterium Helicobacter pylori (B cell gastric lymphoma). Other medically relevant microorganisms that may serve as antigens in mammals and more particularly humans are described extensively in the literature, e.g., C. G. A Thomas, Medical Microbiology, Bailliere Tindall, (1983).

In addition to the above, the methods of the invention can be used for viral infections. Exemplary viral pathogens include, but are not limited to, infectious virus that infect mammals, and more particularly humans. Examples of infectious virus include, but are not limited to: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-I (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g. coronaviruses such as the SARS coronavirus); Rhabdoviradae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses); Bir-naviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; P. oxyiridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g. the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class I=interaally transmitted; class 2=parenterally transmitted (i.e. Hepatitis C); Norwalk and related viruses, and astro viruses).

In addition to the above, the methods of the invention can be used to target gram negative and gram positive bacteria in vertebrate animals. Such gram positive bacteria include, but are not limited to Pasteurella sp., Staphylococci sp., and Streptococcus sp. Gram negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas sp., and Salmonella sp. Specific examples of infectious bacteria include but are not limited to: Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria sp. (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus infuenzae, Bacillus antracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces israelii.

In addition to the above, the methods of the invention can be used to target pathogens that include, but are not limited to, infectious fungi and parasites that infect mammals, and more particularly humans. Examples of infectious fungi include, but are not limited to: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.

In addition to the above, the methods of the invention can be used to target parasites such as intracellular parasites and obligate intracellular parasites. Examples of parasites include but are not limited to Plasmodium-falciparum, Plasmodium ovale, Plasmodium malariae, Plasmdodium vivax, Plasmodium knowlesi, Babesia microti, Babesia divergens, Trypanosoma cruzi, Toxoplasma gondii, Trichinella spiralis, Leishmania major, Leishmania donovani, Leishmania braziliensis, Leishmania tropica, Trypanosoma gambiense, Trypanosoma rhodesiense, Wuchereria bancrofti, Brugia malayi, Brugia timori, Ascaris lumbricoides, Onchocerca volvulus and Schistosoma mansoni.

If modified induced dendritic cells can be used to induce a tolerogenic response including the suppression of a future or existing immune response, to one or more target antigens. Thus, induce DCs are useful for treating or preventing an undesirable immune response including, for example, transplant rejection, graft versus host disease, allergies, parasitic diseases, inflammatory diseases and autoimmune diseases. Examples of transplant rejection, which can be treated or prevented in accordance with the present invention, include rejections associated with transplantation of bone marrow and of organs such as heart, liver, pancreas, kidney, lung, eye, skin etc. Examples of allergies include seasonal respiratory allergies; allergy to aeroallergens such as hayfever; allergy treatable by reducing serum IgE and eosinophilia; asthma; eczema; animal allergies, food allergies; latex allergies; dermatitis; or allergies treatable by allergic desensitisation. Autoimmune diseases that can be treated or prevented by the present invention include, for example, psoriasis, systemic lupus erythematosus, myasthenia gravis, stiff-man syndrome, thyroiditis, Sydenham chorea, rheumatoid arthritis, diabetes and multiple sclerosis. Examples of inflammatory disease include Crohn's disease, chronic inflammatory eye diseases, chronic inflammatory lung diseases and chronic inflammatory liver diseases, autoimmune haemolytic anaemia, idiopathic leucopoenia, ulcerative colitis, dermatomyositis, scleroderma, mixed connective tissue disease, irritable bowel syndrome, systemic lupus erythromatosus (SLE), multiple sclerosis, myasthenia gravis, Guillan-Barre syndrome (antiphospholipid syndrome), primary myxoedema, thyrotoxicosis, pernicious anaemia, autoimmune atrophic gastris, Addison's disease, insulin-dependent diabetes mellitus (IDDM), Goodpasture's syndrome, Behcet's syndrome, Sjogren's syndrome, rheumatoid arthritis, sympathetic ophthalmia, Hashimoto's disease/hypothyroiditis, celiac disease/dermatitis herpetiformis, and demyelinating disease primary biliary cirrhosis, mixed connective tissue disease, chronic active hepatitis, Graves' disease/hyperthyroiditis, scleroderma, chronic idiopathic thrombocytopenic purpura, diabetic neuropathy and septic shock.

Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. The solution is preferably sterile and fluid. Preferably, prior to the introduction of cells, the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

It is preferred that the mode of cell administration is relatively non-invasive, for example by intravenous injection, pulmonary delivery through inhalation, topical, or intranasal administration. However, the route of cell administration will depend on the tissue to be treated and may include implantation. Methods for cell delivery are known to those of skill in the art and can be extrapolated by one skilled in the art of medicine for use with the methods and compositions described herein.

Also provided herein, in some aspects, are kits for making induced dendritic cells (iDCs), the kits comprising any of the DC inducing compositions comprising one or more expression vector components described herein.

Also provided herein, in some aspects, are kits comprising one or more of the DC inducing factors described herein as components for the methods of making the induced dendritic cells described herein.

Accordingly, in some aspects, provided herein, are kits for preparing induced dendritic cells comprising the following components: (a) one or more expression vectors encoding at least one, two, three, four, five, six, seven, eight, or more DC inducing factors selected from: BATF3 (SEQ. ID. 1, SEQ. ID. 2), SPIB (SEQ. ID. 3, SEQ. ID. 4), IRF8 (SEQ. ID. 5, SEQ. ID. 6), PU.1 (SEQ. ID. 7, SEQ. ID. 8), STAT3 (SEQ. ID. 11, SEQ. ID. 12), TCF4 (SEQ. ID. 13, SEQ. ID. 14), IKZF1 (SEQ. ID. 15, SEQ. ID. 16), ID2 (SEQ. ID. 17, SEQ. ID. 18), BCL11A (SEQ. ID. 19, SEQ. ID. 20), RELB (SEQ. ID. 21, SEQ. ID. 22), ZBTB46 (SEQ. ID. 23, SEQ. ID. 24), RUNX3 (SEQ. ID. 25, SEQ: ID. 26), GFI1 (SEQ. ID. 27, SEQ. ID. 28), IRF2 (SEQ. ID. 29, SEQ. ID. 30), NFIL3 (SEQ. ID. 31, SEQ. ID. 32), BCL6 (SEQ. ID. 33, SEQ. ID. 34), L-MYC (SEQ. ID. 35, SEQ. ID. 36), NR4A3 (SEQ. ID. 37, SEQ. ID. 38), and (b) packaging and instructions therefor.

The kits described herein, in some embodiments, can further provide the synthetic mRNAs or the one or more expression vectors encoding DC inducing factors in an admixture or as separate aliquots.

In some embodiments, the kits can further comprise an agent to enhance efficiency of reprogramming. In some embodiments, the kits can further comprise one or more antibodies or primer reagents to detect a cell-type specific marker to identify cells induced to the dendritic cell state.

In some embodiments, the kits can further comprise a buffer. In some such embodiments, the buffer is RNase-free TE buffer at pH 7.0. In some embodiments, the kit further comprises a container with cell culture medium.

All kits described herein can further comprise a buffer, a cell culture medium, a transduction or transfection medium and/or a media supplement. In preferred embodiments, the buffers, cell culture mediums, transfection mediums, and/or media supplements are DNAse and RNase-free. In some embodiments, the synthetic, modified RNAs provided in the kits can be in a non-solution form of specific quantity or mass, e.g., 20 μg, such as a lyophilized powder form, such that the end-user adds a suitable amount of buffer or medium to bring the components to a desired concentration, e.g., 100 ng/μl.

All kits described herein can further comprise devices to facilitate single-administration or repeated or frequent infusions of the cells generated using the kits components described herein, such as a non-implantable delivery device, e.g., needle, syringe, pen device, or an implantatable delivery device, e.g., a pump, a semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or a reservoir. In some such embodiments, the delivery device can include a mechanism to dispense a unit dose of a pharmaceutical composition comprising the iDCs. In some embodiments, the device releases the composition continuously, e.g., by diffusion. In some embodiments, the device can include a sensor that monitors a parameter within a subject. For example, the device can include pump, e.g., and, optionally, associated electronics.

In an embodiment, induced dendritic cells are made by the hand of man by, e.g., modifying the gene expression of at least one of the factors disclosed herein of a somatic cell, a pluripotent cell, a progenitor cell or a stem cell, or by exposing any one of these cell types to at least one protein or RNA that produces at least one protein as disclosed herein. The cells can further be made by exposing them to small molecules that turn on at least one of the factors disclosed herein. In some aspects at least two, three, four, five, six, seven, or eight factors are used to make the induced dendritic cells.

In an embodiment, the induced dendritic cells in some aspects of all the embodiments of disclosure, while similar in functional characteristics, differ significantly in their gene expression from the naturally occurring endogenous dendritic cells.

In an embodiment, the induced dendritic cells as described herein differ from naturally occurring dendritic cells by both their posttranslational modification signatures and their gene expression signatures.

In an embodiment, the induced dendritic cells as described herein differ from naturally occurring dendritic cells by their ability to growth in vitro as adherent cultures and to survive in culture for more than one month.

In an embodiment, induced dendritic cell is also defined as comprising a gene expression signature that differs from naturally occurring dendritic cells. One can experimentally show the difference by comparing the gene expression pattern of a naturally occurring dendritic cell to that of the induced dendritic cells. Therefore, in some aspects of all the embodiments of the invention, the induced dendritic cells comprise an expression signature that is about 1-5%, 5-10%, 5-15%, or 5-20% different from the expression signature of about 1-5%, 2-5%, 3-5%, up to 50%, up to 40%, up to 30%, up to 25%, up to 20%, up to 15%, or up to 10% of specific genes. For example, expression levels of DC inducing factor(s), such as PU.1, IRF8, BATF3 and TCF4, in iDCs are higher than in naturally occurring DCs as the DC inducing factors are being overexpressed.

In an embodiment, mouse Embryonic Fibroblasts (MEFs) were isolated and purified in the following way: Clec9aCre/Cre animals (10) were crossed with Rosa26-stopflox-tdTomato reporter mice (The Jackson Laboratory) to generate double homozygous Clec9a^(Cre/Cre) Rosa^(tdTomato/tdTomato) (C9A-tdTomato) mice. C57BL/6 mice, Rag2 constitutive knock-out (KO)/OT-II random transgenic (Rag2KO/OT-II) mice and Rag2KO/OT-I random transgenic mice were acquired from Charles River and Taconic, respectively (17-19). All animals were housed under controlled temperature (23±2° C.), subject to a fixed 12-h light/dark cycle, with free access to food and water.

In an embodiment, primary cultures of MEFs were isolated from E13.5 embryos of C9A-tdTomato or C57BL/6 mice (6, 10). Head, fetal liver and all internal organs were removed and the remaining tissue was mechanically dissociated. Dissected tissue was enzymatic digested using 0.12% trypsin/0.1 mM Ethylenediaminetetraacetic acid (EDTA) solution (3 mL per embryo), and incubation at 37° C. for 15 min. Additional 3 mL of same solution per embryo were added, followed by another 15 min incubation period. A single cell suspension was obtained and plated in 0.1% gelatin-coated 10-cm tissue culture dishes in growth media. Cells were grown for 2-3 days until confluence, dissociated with Tryple Express and frozen in Fetal Bovine Serum (FBS) 10% dimethyl sulfoxide (DMSO). Before plating for lentiviral transduction, MEFs were sorted to remove residual CD45+ and tdTomato+ cells that could represent cells with hematopoietic potential. MEFs used for screening and in the following experiments were tdTomato-CD45− with a purity of 99.8% and expanded up to 4 passages.

In an embodiment, HEK293T cells, MEFs and Human Dermal Fibroblasts (HDFs, ScienCell) were maintained in growth medium [Dulbecco's modified eagle medium (DMEM) supplemented with 10% (v/v) FBS, 2 mM L-Glutamine and antibiotics (10 μg/ml Penicillin and Streptomycin)], OP-9 and OP-9-DL1 cell lines were cultured in Minimum Essential Medium (MEM) Alpha containing 20% FBS, 1 mM L-Glutamine and penicillin/streptomycin (10 μg/ml). OP-9 and OP-9-DL1 were routinely passaged at 80% confluency. All cells were maintained at 37° C. and 5% (v/v) CO2. All tissue culture reagents were from Thermo Fisher Scientific unless stated otherwise.

In an embodiment, viral transduction and reprogramming experiments were performed in the following way: C9A-tdTomato MEFs were seeded at a density of 40,000 cells per well on 0.1% gelatin coated 6-well plates. Cells were incubated overnight with a ratio of 1:1 FUW-TetO-TFs and FUW-M2rtTA lentiviral particles in growth media supplemented with 8 μg/mL polybrene. When testing combinations of TFs, equal MOIs of each individual viral particles were applied. Cells were transduced twice in consecutive days and after overnight incubation, media was replaced with fresh growth media. After the second transduction, growth media was supplemented with Doxycycline (1 μg/mL)—day 0. Media was changed every 2-3 days for the duration of the cultures. Emerging tdTomato+ cells were analyzed 1-15 days post-transduction. When stated, variations of culture conditions were applied, namely RPMI-1640, Lipopolysaccharide (LPS, 100 ng/ml, Sigma), 2-Mercaptoethanol (1×104 μM; 2-ME), L-glutamine (2 μmol/ml), GM-CSF (10 ng/ml, STEMCELL Technologies), IL-4 (20 ng/ml, STEMCELL Technologies) and Flt3I (100 ng/ml, STEMCELL Technologies).

In an embodiment, fluorescent microscopy and immunofluorescence was evaluated in the following way: C9A-driven tdTomato in MEFs and transduced HDFs were visualized directly on 6-well plates under an inverted microscope (Zeiss AxioVert 200M) and images processed with AxioVision and Adobe Photoshop software. DAPI (4′,6-diamidino-2-phenylindole, 1 μg/mL, Sigma) and Phalloidin (50 μg/ml, Sigma) were used to stain nuclei and F-actin, respectively. For time-lapse microscopy fluorescent pictures were acquired after adding Dox every 1 hour for 6 days and 4 hours using an INCELL Analyzer 2200 (GE Healthcare). Movies were generated with Image) software (NIH).

In an embodiment, flow cytometry analysis was performed in the following way: Transduced C9A-tdTomato MEFs or transduced human fibroblasts were dissociated with TrypLE Express, resuspended in 200 μL PBS 5% FBS and kept at 4° C. prior analysis in BD Accuri C6 Flow Cytometer (BD Biosciences). Sample acquisition was performed with the configuration 3-blue-1-red (533/30 filter in FL1; 585/40 in FL2, 670 LP in FL3 and 675/25 in FL4). tdTomato fluorescence was analyzed in the FL2 channel. For the analysis of CD45 or MHC-II cell surface marker expression, dissociated cells were incubated with APC-Cy7 rat anti-mouse CD45 antibody or Alexa Fluor 647 rat anti-mouse I-A/I-E diluted in PBS 5% FBS at 4° C. for 30 minutes in the presence of rat serum (1/100, GeneTex) to block unspecific binding. Cells were washed with PBS 5% FBS, resuspended in PBS 5% FBS and analyzed in a BD Accuri C6 Flow cytometer. CD45 APC-Cy7 and I-A/I-E Alexa Fluor 647 fluorescence were analyzed in FL4 channel. For the combined analysis of MHCII, CD80 and CD86 cell surface expression, dissociated cells were stained with Alexa Fluor 647 rat anti-mouse I-A/I-E, BV650 rat anti-mouse CD80 and PE-CY7 rat anti-mouse CD86 and analyzed in BD FACSAria III (BD Biosciences). For the analysis of transduced HDFs, dissociated cells were stained with APC mouse anti-human CLEC9A and FITC mouse anti-human HLA-DR. To assess CD4+ and CD8+ T cell proliferation and activation after 7 days of co-culture with APCs, carboxyfluorescein succinimidyl ester (CFSE)-labeled T cells were incubated with PE rat anti-mouse CD44 and analyzed in BD Accuri C6. Flow cytometry data were analyzed using FlowJo software (FLOWJO, LLC, version 7.6).

In an embodiment, fluorescence activated cell sorting (FACS) was performed in the following way: To purify C9A-tdTomato MEFs, cells were incubated at 4° C. for 30 minutes with APC-Cy7 anti-CD45 antibody diluted in PBS 5% FBS. Subsequently, MEFs were washed with PBS 5% FBS, resuspended in PBS 5% FBS and tdTomato− CD45− MEFs were purified in BD FACSAria III. When described tdTomato+ cells were purified using BD FACSAria III and cultured in the absence or presence of doxycycline. For the isolation of splenic DCs, splenic cells were incubated with Alexa Fluor 647 rat anti-mouse I-A/I-E, FITC rat anti-mouse CD11c and APC-Cy7 rat anti-mouse CD8a. CD11c+MHCII+CD8α+ splenic DCs were purified in BD FACSAria III (BD Biosciences). FACS data was processed in FlowJo software.

In an embodiment, GPSforGenes software was used to calculate the specificity of Pu.1, Irf8 and Batf3 combination for the DC lineage. Gene expression data was downloaded from BioGPS database (GeneAtlas MOE430), transformed to log-space and normalized to bring the expression values to 0-1 range for each gene across different samples. The resulting data was then searched for samples with the highest averaged expression for Pu.1+Irf8+Batf3.

In an embodiment, Single cell mRNAseq analysis was performed in the following way: Single-end reads were mapped to the mm10 mouse genome (Ensembl annotation, release 89) using Salmon v0.8.1 with k=21. The resulting TPM were imported into R using tximport library and converted into mRNA counts using the Census algorithm implemented in monocle library. Scatter library was used to discard cells and genes that didn't pass quality control threshold. The following QC criteria were used: 1) library size per cell; 2) number of genes detected in each single cell; 3) percentage of counts in mitochondrial genes. From the 192 cells initially profiled, 163 individual cells passed quality control filters and were used for analysis. Custom R scripts were used to perform t-distributed stochastic neighbor embedding (tSNE) (Monocle and scatter package), principal component analysis (PCA) (Monocle and scatter package), hierarchical clustering (SC3 package), variance analysis and to construct heat maps, box plots, scatter plots, violin plots, dendrograms, bar graphs, and histograms. Generally, ggplot2, gplots, graphics and pheatmap packages were used to generate data graphs.

In an embodiment, differential expression analysis was performed using Monocle package, and selecting genes with BH-corrected p-value less than 0.05. The resulting genes were next filtered by variance (genes with variance >=1 across all conditions were selected). Finally, the resulting 6,525 genes were grouped into 4 distinct clusters based on hierarchical clustering.

In an embodiment, endogenous expression of genes was determined using STAR v2.5.3a with default settings. A window was defined based on −10 kb, start of the gene and end of the gene, +10 kb, which correspond to 5′ and 3′ untranslated regions (UTRs) and used to calculate the number of reads in the UTRs using multicov from bedtools v2.27.0.

In an embodiment, DC lineage of iDCs was determined by using cDC1 and cDC2 gene signatures from Schlitzer (11). The majority of genes were highly expressed in MEF, and across all our condition. These genes were discarded. Moreover, as sDC cells were purified for the cDC1 markers CD11c, MHC-II and CD8α, genes that were expressed in sDC but at the same time were found in cDC2 signature list were discarded. cDC1/cDC2 gene lists were then used to performed hierarchical clustering. Next, only clusters in which median expression of genes in MEF cells were significantly lower compared to day 3, day 7 and day 9 were selected. Besides that, for cDC2 gene list, in addition to procedure described above, gene clusters with median expression of genes in sDC cells significantly higher compared to day 3, day 7 and day 9 were also discarted. Next, the median of gene expression across each selected gene was calculated from each particular condition. Finally, the median of gene expression across all pre-sorted cDC1 and cDC2 gene signatures defined by Schlitzer and colleagues, was calculated.

In an embodiment, the Monocle package, an algorithm that uses independent component analysis with minimal spanning tree to connect cells along a pseudotemporally ordered path, was used to order cells on a pseudo-time course during MEF to iDC cell reprogramming. Monocle analysis was performed based on cDC1 and cDC2 genes from Schlitzer, 2015 (11) as genes, which define a cell's progress, as this was an alternative approach to prove that day 9 are cDC1-like cells. The resulting trajectories were visualized using Monocle functions. Since single-cell trajectories included branches, branched expression analysis modeling (BEAM) was used, a special statistical test implemented in Monocle package in order to find differentially expressed genes between the branches. As an alternative approach to Monocle branching algorithm, TSCAN was used, which combines clustering with pseudotime analysis, by building a minimum-spanning tree to connect the clusters. TSCAN, in contrast to Monocle, can use all genes to order the cells.

In an embodiment, gene ontology (biological process, cellular component and KEGG pathway) was performed using Enrichr (amp.pharm.mssm.edu/Enrichr/) and Database for Annotation, Visualization and Integrated Discovery (DAVID) clustered functional analysis (david.ncifcrf.gov/).

In an embodiment, microRNA target interaction analysis was performed using miRTarBase 2017, Enrichr website (amp.pharm.mssm.edu/Enrichr/).

In an embodiment, Mouse phenotype analysis was performed using Network2canvas (www.maayanlab.net/N2C/#.WmRvOjLc8yk).

In an embodiment, gene set enrichment analysis (GSEA) between all possible conditions and states were performed against C7: immunologic signatures from Molecular Signatures Database (MSigDB) and NetPath.

In an embodiment, TF network analysis was computed by pairwise correlation matrix using Pearson correlation. TFs were selected based on DBD: Transcription factor prediction database (http://www.transcriptionfactor.org/) in mouse. As the objective was to investigate the switch between condition from mef to day 9, 3 lists were created corresponding to switch from mef to day 3; day 3 to day 7; day 7 to day 9 and included only those TF which have log FC=0.5 for pair of conditions. Next, out of TFs defined based on log FC for 3 pair of conditions TFs were selected with a Pearson correlation of greater than 0.35 with at least five other TFs. Taking into consideration the fact that those results could be obtained by chance, permutations were used in order to determine the probability of TFs passing this threshold by chance. 100 permutations were performed and all of them resulted in 0 TFs that pass this threshold. The function graph.adjacency( ) of igraph R package was used, which took Pearson pairwise correlation matrix for the selected TFs for 3 pair of conditions.

In an embodiment, Methylcelulose clonogenic assays were performed in the fallowing way: PIB-transduced MEFs at day 3, 5, 7, 10 and 25 after addition of Dox were assayed in 1% methylcellulose media (Methocult M3434, Stem Cell Technologies). Sorted sDC1 (MHC-II+CD11c+CD8a+) as well as unsorted splenocytes and bone marrow cells were used as control. Hematopoietic colonies were scored and counted after 7-10 days of culture in 5% CO2 at 37° C.

In an embodiment, bead incorporation assay was evaluated in the following way: transduced C9A-tdTomato MEF or transduced HDF cultures were incubated with 2.5% yellow-green fluorescent-coupled solid latex beads (carboxylate-modified polystyrene, Sigma) at 1:1000 ratio in growth medium. Sixteen hours later, cells were washed twice in PBS 5% FBS and analyzed under an inverted microscope. DAPI (1 μg/mL, Sigma) was used for nuclear staining.

In an embodiment, incorporation of labelled ovalbumin was evaluated in the following way: transduced MEFs and human fibroblast cultures were incubated with Alexa647-labelled ovalbumin (Life Technologies) for 20 minutes at 37° C. or 4° C. After washing with PBS 5% FBS, cells were analysed in BD Accuri C6.

In an embodiment, incorporation of dead cells was evaluated in the following way: HEK293T cells were exposed to ultra-violet (UV) irradiation to induce cell death and labelled with CellVue® Claret Far Red Fluorescent Cell Linker Kit (Sigma), according to manufacturer's instructions. Transduced MEFs were incubated with Far red-labelled dead cells overnight, and analysed in BD Accuri C6.

In an embodiment, inflammatory cytokine assay was performed in the following way: Levels of the cytokines interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), interferon-γ (IFN-γ) and tumor necrosis factor (TNF) were assessed in supernatants of iDCs cultures 10 days after Dox supplementation. At day 9, 100 ng/mL LPS or 25 μg/mL of Polyinosinic-polycytidylic acid (PolyI:C) (Invivogen) were added for overnight stimulation. 50 μL of culture supernatants from a 6-well plate well were collected and analyzed by CBA Mouse Inflammation Kit (BD Biosciences), according to manufacturer's instructions. Acquisition was performed with a BD Accuri C6 and data were analyzed using FCAP array software, version 3.0 (BD Biosciences). The limit of detection in CBA was: IL-6, 20.91 pg/ml; IL-10, 10.55 pg/ml; IFN-γ, 18.2 pg/ml; TNF, 18.13 pg/ml; IL-12p70, 20.05 pg/ml.

In an embodiment, splenic DC isolation was evaluated in the following way: Freshly isolated spleens were homogenized using the frosted ends of 2 sterile slides. Cells were harvested in PBS supplemented with 2% FBS and filtered through a 70 μm cell strainers (BD Biosciences). Red blood cells were lysed with BD Pharm Lyse (BD Biosciences) for 8 min at room temperature. MHC-II+CD11c+ DCs were purified by FACS (BD FACSAria III, BD Biosciences) and immediately used for antigen presenting assays.

In an embodiment, CD4+ T cell isolation and antigen presenting assays was evaluated in the following way: CD4+ T cells from spleen of Rag2KO/OT-11 mice were enriched using Dynabeads Untouched Mouse CD4 Cells Kit (BD Biosciences), according to manufacturer's instructions. Enriched CD4+ T cells were labeled with CFSE 5 μM at room temperature for 10 min, washed, and counted before cultured with APCs. iDCs cultures at day 8 after the addition of Dox or splenic CD11c+ MHC-II+ DCs cells were incubated with OVA protein (10 μg/mL) or OVA323-339 peptide (10 μg/mL) in the presence or absence of 100 ng/mL of LPS and co-cultured with untouched CFSE-labeled OT-II CD4+ T cells. iDC cultures (20000 cells) or 20000 splenic CD11c+ MHC-II+ DCs were incubated with 20000 CFSE-labeled CD4+ T cells in 96-well round-bottom tissue culture plates. T cell proliferation (dilution of CFSE staining) and activation (CD44 expression) were assessed by flow cytometry after 7 days of co-culture.

In an embodiment, CD8+ T cell isolation and antigen cross-presentation was evaluated in the following way: CD8+ T cells from spleen of Rag2KO/IT-1 mice were enriched using Dynabeads Untouched Mouse CD8 Cells Kit (BD Biosciences), according to manufacturer's instructions. Enriched CD8+ T cells were labelled with CFSE 5 μM at room temperature for 10 min, washed, and counted before cultured with APCs. iDCs cultures at day 8 after the addition of Dox or splenic CD11c+ MHC-II+ DCs cells were incubated with OVA protein (10 μg/mL) in the presence of 25 μg/mL of polyI:C and co-cultured with untouched CFSE-labelled OT-1 CD8+ T cells. iDC cultures (20000 cells) or 20000 splenic CD11c+ MHC-II+ DCs were incubated with 20000 CFSE-labelled CD8+ T cells in 96-well round-bottom tissue culture plates. T cell proliferation (dilution of CFSE staining) and activation (CD44 expression) were assessed by flow cytometry after 4 days of co-culture.

In an embodiment, hybridoma cross-presentation assays were performed in the fallowing way: PIB-transduced Clec9a-tdTomato MEFs at day 16 after addition of Dox were dissociated with TrypLE Express, resuspended in growth media and incubated for 4 hours with different concentrations of OVA protein. After being extensively washed, PIB-transduced MEFs (100,000 cells) were co-cultured with 100,000 B3Z cells in 96-well round-bottom tissue culture plates in the presence or absence of 100 ng/mL LPS or 25 μg/mL PIC. After 18 h, cells were lysed in a buffer containing 0.125% Nonidet P-40 (substitute), 9 mM MgCl2, and a colorimetric CPRG β-galactosidase substrate. β-galactosidase activity was measured on MicroPlate Reader as optical density at 590 nm.

In an embodiment, the efficiency of antigen export to the cytosol by Clec9a− tdTomato+ cells were analyzed by cytofluorimetry-based assay. Briefly, PIB-transduced MEFs at day 16 after addition of Dox were dissociated with TrypLE Express, resuspended in loading buffer and loaded with 1 μM CCF4-AM for 30 min at room temperature. Cells were then washed and incubated with 2 mg/mL β-lactamase at 37° C. for 30, 60 and 90 minutes. To stop the reaction, cells were transferred to ice cold PBS. Immediately before flow cytometry analysis in a BD FACSAriaIII, the cells were stained with Fixable Viability Dye eFluor 780 (eBioscience). The percentage of live Clec9a-tdTomato+ cells with a high blue-to-green (V450/V500) fluorescence ratio was used as a measure of the efficiency of antigen export into the cytosol.

In an embodiment, comparisons among groups were performed by one-way ANOVA followed by Bonferroni's multiple comparison test with GraphPad Prism 5 software. P-values are shown when relevant (*p<0.05; **p<0.01, ***p<0.001, ****p<0.0001).

TABLE 6 Primary Antibodies Used in the analysis Antibody/ Antigen Specie Clone Conjugate Source CD45 Mouse 30-F11 PE BD Pharmingen CD4 Mouse GK1.5 PE-CY7 eBioscience CD8α Mouse 53-6.7 APC-Cy7 Biolegend CD44 Mouse IM7 PE BD Pharmingen CD103 Mouse 2E7 APC-Cy7 Biolegend MHC Class II Mouse M5/114.15.2 Alexa Fluor 647 BD Pharmingen (I-A/I-E) MHC Class I Mouse AF6-88.5.5.3 FITC eBioscience (H-2Kb) CD80 (B7 1) Mouse 16-101A BV650 BD Horizon CD86 (B7 2) Mouse GL1 PE-CY7 eBioscience CD40 Mouse HM40-3 eFluor450 eBioscience B220 Mouse RA3-6B2 FITC eBioscience CD11b Mouse M1/70 AlexaFluor700 eBioscience CD11c Mouse N418 FITC Biolegend HLA-DR Human L243 FITC Biolegend CLEC9A Human 8F9 APC Biolegend

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims.

Where singular forms of elements or features are used in the specification of the claims, the plural form is also included, and vice versa, if not specifically excluded. For example, the term “a cell” or “the cell” also includes the plural forms “cells” or “the cells,” and vice versa. In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

The above described embodiments are combinable.

The following claims further set out particular embodiments of the present disclosure.

All references recited in this document are incorporated herein in their entirety by reference, as if each and every reference had been incorporated by reference individually.

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1.-49. (canceled)
 50. A construct or vector comprising a combination of at least two polynucleotide sequences encoding at least two transcription factors selected from the group consisting of: BATF3, IRF8 and PU.1.
 51. The construct or vector according to claim 50, wherein the at least two transcription factors are encoded by a sequence at least 90% identical to a sequence selected from the group consisting of: i. SEQ. ID. 1 or SEQ. ID. 2 (BATF3), ii. SEQ. ID. 5 or SEQ. ID. 6 (IRF8), and iii. SEQ. ID. 7 or SEQ. ID. 8 (PU.1).
 52. The construct or vector according to claim 50, wherein the at least two transcription factors are encoded by a sequence at least 95%, identical to a sequence selected from the group consisting of: i. SEQ. ID. 1 or SEQ. ID. 2 (BATF3), ii. SEQ. ID. 5 or SEQ. ID. 6 (IRF8), and iii. SEQ. ID. 7 or SEQ. ID. 8 (PU.1).
 53. The construct or vector according to claim 50, wherein the at least two transcription factors are BATF3 and PU.1, or IRF8 and PU.1.
 54. The construct or vector according to claim 50, wherein the construct comprises at least three transcription factors, wherein said three transcription factors are in the following sequential order from 5′ to 3′: PU.1, IRF8, BATF3; or IRF8, PU.1, BATF3.
 55. The construct or vector according to claim 50, wherein the vector is a viral vector.
 56. The construct or vector according to claim 55, wherein the viral vector is selected from the group consisting of: lentiviral, adeno-associated viral, adenoviral, retroviral, herpes viral, and pox viral vector.
 57. The construct or vector according to claim 50, wherein the transcription factors sequences are operably linked to a promoter region capable of controlling the transcription of said transcription factors.
 58. The construct or vector according to claim 57, wherein the promoter region comprises the tetracycline operator and a minimal cytomegalovirus (CMV) promoter or the constitutively active human ubiquitin C promoter (UbC).
 59. A pharmaceutical composition comprising a combination of at least two isolated transcription factors encoded by a sequence at least 90% identical to a sequence selected from a list consisting of: BATF3 (SEQ. ID. 1 or SEQ. ID. 2), IRF8 (SEQ. ID. 5 or SEQ. ID. 6), PU.1 (SEQ. ID. 7 or SEQ. ID. 8), and mixtures thereof, and one or more pharmaceutically acceptable excipients and/or carriers.
 60. The pharmaceutical composition according to claim 59 comprising a combination of at least two isolated transcription factors encoded by a sequence at least 95% identical to a sequence from a list consisting of selected from a list consisting of: BATF3 (SEQ. ID. 1 or SEQ. ID. 2), IRF8 (SEQ. ID. 5 or SEQ. ID. 6), PU.1 (SEQ. ID. 7 or SEQ. ID. 8), and mixtures thereof.
 61. The pharmaceutical composition according to claim 59 wherein the combination of isolated transcription factors is selected from the following encoded combinations: BATF3 (SEQ. ID. 1 or SEQ. ID. 2), IRF8 (SEQ. ID. 5 or SEQ. ID. 6) and PU.1 (SEQ. ID. 7 or SEQ. ID. 8); or BATF3 (SEQ. ID. 1 or SEQ. ID. 2) and IRF8 (SEQ. ID. 5 or SEQ. ID. 6); or IRF8 (SEQ. ID. 5 or SEQ. ID. 6) and PU.1 (SEQ. ID. 7 or SEQ. ID. 8); or BATF3 (SEQ. ID. 1 or SEQ. ID. 2) and PU.1 (SEQ. ID. 7 or SEQ. ID. 8).
 62. The pharmaceutical composition according to claim 59, wherein the combination of isolated transcription factors is the encoded combination of BATF3 (SEQ. ID. 1 or SEQ. ID. 2), IRF8 (SEQ. ID. 5 or SEQ. ID. 6) and PU.1 (SEQ. ID. 7 or SEQ. ID. 8).
 63. The pharmaceutical composition according to claim 59, further comprising at least one polynucleotide encoding an agent selected from the group consisting of: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-18, IL-19, IL-20, IFN-α, IFN-β, IFN-γ, TNF, TGF-β, G-CSF, M-CSF, GM-CSF, Flt-3 ligand, kit ligand, and mixtures thereof.
 64. A method of treatment of cancer, the method comprising administering to a patient in need thereof the construct or vector according to claim
 50. 65. The method of treatment according to claim 64, wherein the cancer is selected from the group consisting of: benign tumor, malignant tumor, early cancer, basal cell carcinoma, cervical dysplasia, soft tissue sarcoma, germ cell tumor, retinoblastoma, Hodgkin's lymphoma, blood cancer, prostate cancer, ovarian cancer, cervix cancer, uterus cancer, vaginal cancer, breast cancer, naso-pharynx cancer, trachea cancer, larynx cancer, bronchi cancer, bronchioles cancer, lung cancer, hollow organs cancer, esophagus cancer, stomach cancer, bile duct cancer, intestine cancer, colon cancer, colorectal cancer, rectum cancer, bladder cancer, ureter cancer, kidney cancer, liver cancer, gall bladder cancer, spleen cancer, brain cancer, lymphatic system cancer, bone cancer, pancreatic cancer, leukemia, skin cancer, and myeloma. 